Antibodies to CD40

ABSTRACT

The present invention relates to antibodies and antigen-binding portions thereof that specifically bind to CD40, preferably human CD40, and that function as CD40 agonists. The invention also relates to human anti-CD40 antibodies and antigen-binding portions thereof. The invention also relates to antibodies that are chimeric, bispecific, derivatized, single chain antibodies or portions of fusion proteins. The invention also relates to isolated heavy and light chain immunoglobulins derived from human anti-CD40 antibodies and nucleic acid molecules encoding such immunoglobulins. The present invention also relates to methods of making human anti-CD40 antibodies, compositions comprising these antibodies and methods of using the antibodies and compositions for diagnosis and treatment. The invention also provides gene therapy methods using nucleic acid molecules encoding the heavy and/or light immunoglobulin molecules that comprise the human anti-CD40 antibodies. The invention also relates to transgenic animals comprising nucleic acid molecules of the present invention.

This application claims the benefit of U.S. Provisional Application60/348,980, filed Nov. 9, 2001.

BACKGROUND OF THE INVENTION

The CD40 antigen is a 50 kDa cell surface glycoprotein which belongs tothe Tumor Necrosis Factor Receptor (TNF-R) family. (Stamenkovic et al.,EMBO J. 8: 1403-10 (1989).) CD40 is expressed in many normal and tumorcell types, including B lymphocytes, dendritic cells, monocytes,macrophages, thymic epithelium, endothelial cells, fibroblasts, andsmooth muscle cells. (Paulie S. et al., Cancer Immunol. Immunother.20:23-8 (1985); Banchereau J. et al., Adv. Exp. Med. & Biol. 378:79-83(1995); Alderson M. R. et al., J. of Exp. Med. 178:669-74 (1993);Ruggiero G. et al., J. of Immunol. 156:3737-46 (1996); Hollenbaugh D. etal., J. of Exp. Med. 182:33-40 (1995); Yellin M. J. et al., J. ofLeukocyte Biol. 58:209-16 (1995); and Lazaar A. L. et al., J. ofImmunol. 161:3120-7 (1998).) CD40 is expressed in all B-lymphomas and in70% of all solid tumors. Although constitutively expressed, CD40 isup-regulated in antigen presenting cells by maturation signals, such asLPS, IL-1βp, IFN-γ and GM-CSF.

CD40 activation plays a critical role in regulating humoral and cellularimmune responses. Antigen presentation without CD40 activation can leadto tolerance, while CD40 signaling can reverse such tolerance, enhanceantigen presentation by all antigen presenting cells (APCs), lead tosecretion of helper cytokines and chemokines, increase co-stimulatorymolecule expression and signaling, and stimulate cytolytic activity ofimmune cells.

CD40 plays a critical role in B cell proliferation, maturation and classswitching. (Foy T. M. et al., Ann. Rev. of Immunol. 14:591-617 (1996).)Disruption of the CD40 signaling pathway leads to abnormal serumimmunoglobulin isotype distribution, lack of CD4+ T cell priming, anddefects in secondary humoral responses. For example, the X-linkedhyper-IgM syndrome is a disease associated with a mutation in the humanCD40L gene, and it is characterized by the inability of affectedindividuals to produce antibodies other than those of the IgM isotype,indicating that the productive interaction between CD40 and CD40L isrequired for an effective immune response.

CD40 engagement by CD40L leads to the association of the CD40cytoplasmic domain with TRAFs (TNF-R associated factors). (Lee H. H. etal., Proc. Natl. Acad. Sci. USA 96:1421-6 (1999); Pullen S. S. et al.,Biochemistry 37:11836-45 (1998); Grammar A. C. et al., J. of Immunol.161:1183-93 (1998); Ishida T. K. et al., Proc. Natl. Acad. Sci. USA93:9437-42 (1996); Pullen S. S. et al., J. of Biol. Chem. 274:14246-54(1999)). The interaction with TRAFs can culminate in the activation ofboth NFκB and Jun/AP1 pathways. (Tsukamoto N. et al., Proc. Natl. Acad.Sci. USA 96:1234-9 (1999); Sutherland C. L. et al., J. of Immunol.162:4720-30 (1999).) Depending on cell type, this signaling leads toenhanced secretion of cytokines such as IL-6 (Jeppson J. D. et al., J.of Immunol. 161:1738-42 (1998); Uejima Y. et al., Int. Arch. of Allergy& Immunol. 110:225-32, (1996), IL-8 (Gruss H. J. et al., Blood84:2305-14 (1994); von Leoprechting A. et al., Cancer Res. 59:1287-94(1999); Denfeld R. W. et al., Europ. J. of Immunol. 26:2329-34 (1996)),IL-12 (Cella M. et al., J. of Exp. Med. 184:747-52 (1996); Ferlin W. G.et al., Europ. J. of Immunol. 28:525-31 (1998); Armant M. et al., Europ.J. of Immunol. 26:1430-4 (1996); Koch F. et al., J. of Exp. Med.184:741-6 (1996); Seguin R. and L. H. Kasper, J. of Infect. Diseases179:467-74 (1999); Chaussabel D. et al., Infection & Immunity 67:1929-34(1999)), IL-15 (Kuniyoshi J. S. et al., Cellular Immunol. 193:48-58(1999)) and chemokines (MIP1α, MIP1β, RANTES, and others) (McDyer J. F.et al., J. of Immunol. 162:3711-7 (1999); Schaniel C. et al., J. of Exp.Med. 188:451-63 (1998); Altenburg A. et al., J. of Immunol. 162:4140-7(1999); Deckers J. G. et al., J. of the Am. Society of Nephrology9:1187-93 (1998)), increased expression of MHC class I and II(Santos-Argumedo L. et al., Cellular Immunol. 156:272-85 (1994)), andincreased expression of adhesion molecules (e.g., ICAM) (Lee H. H. etal., Proc. Natl. Acad. Sci. USA. 96:1421-6 (1999); Grousson J. et al.,Archives of Dermatol. Res. 290:325-30 (1998); Katada Y. et al., Europ.J. of Immunol. 26:192-200 (1996); Mayumi M. et al., J. of Allergy &Clin. Immunol. 96:1136-44 (1995); Flores-Romo L. et al., Immunol.79:445-51 (1993)) and costimulatory molecules (e.g., B7) (Roy M. et al.,Europ. J. of Immunol. 25:596-603 (1995); Jones K. W. and C. J. Hackett,Cellular Immunol. 174:42-53 (1996); Caux C. et al., Journal of Exp. Med.180:1263-72 (1994); Kiener P. A. et al., J. of Immunol. 155:4917-25(1995)). Cytokines induced by CD40 engagement enhance T cell survivaland activation.

In addition to enhancement of cellular and immune function, the effectsof CD40 activation include: cell recruitment and differentiation bychemokines and cytokines; activation of monocytes; increased cytolyticactivity of cytolytic T lymphocyte (CTL) and natural killer (NK) cells;induction of apoptosis in CD40 positive tumors; enhancement ofimmunogenicity of CD40 positive tumors; and tumor-specific antibodyproduction. The role of CD40 activation in cell-mediated immuneresponses is also well established, and it is reviewed in: Grewal etal., Ann. Rev. of Immunol. 16:111-35 (1998); Mackey et al., J. ofLeukocyte Biol. 63:418-28 (1998); and Noelle R. J., Agents &Actions-Suppl. 49:17-22 (1998). Studies using a cross-priming modelsystem showed that CD40 activation of APCs can replace helper T cellrequirement for the generation of cytolytic T lymphocyte (CTL). (Bennettet al., Nature 393:478-480 (1998).) Evidence from CD40L deficient miceindicates a clear requirement for CD40 signaling in helper T cellpriming. (Grewal I. S. et al., Science 273:1864-7 (1996); Grewal I. S.et al., Nature 378:617-20 (1995).) CD40 activation converts otherwisetolerogenic, antigen bearing B cells into competent APCs. (Buhlmann J.E. et al., Immunity 2:645-53 (1995).) CD40 activation induces maturationand differentiation of cord blood progenitors into dendritic cells.(Flores-Romo L. et al., J. of Exp. Med. 185:341-9 (1997); Mackey M. F.et al., J. of Immunol. 161:2094-8 (1998).) CD40 activation also inducesdifferentiation of monocytes into functional dendritic cells. (BrossartP. et al., Blood 92:4238-47 (1998).) Further, CD40 activation enhancescytolytic activity of NK cells through APC-CD40 induced cytokines.(Carbone E. et al., J. of Exp. Med. 185:2053-60 (1997); Martin-FontechaA. et al., J. of Immunol. 162:5910-6 (1999).) These observationsindicate that CD40 plays an essential role in the initiation andenhancement of immune responses by inducing maturation of APCs,secretion of helper cytokines, upregulation of costimulatory molecules,and enhancement of effector functions.

The critical role of CD40 signaling in the initiation and maturation ofhumoral and cytotoxic immune responses makes this system an ideal targetfor immune enhancement. Such enhancement can be particularly importantfor mounting effective immune responses to tumor antigens, which aregenerally presented to the immune system through cross-priming ofactivated APCs. (Huang A. Y. et al., Ciba Foundation Symp. 187:229-44(1994); Toes R. E. M. et al., Seminars in Immunol. 10:443-8 (1998);Albert M. L. et al., Nature 392:86-9 (1998); Bennett S. R. et al., J. ofExp. Med. 186:65-70 (1997).)

Several groups have demonstrated the effectiveness of CD40 activationfor antitumor responses in vitro and in vivo. (Toes R. E. M. et al.,Seminars in Immunol. 10:443-8 (1998).) Two groups, using lung metastaticmodel of renal cell carcinoma and subcutaneous tumors by virallytransformed cells, have independently demonstrated that CD40 activationcan reverse tolerance to tumor-specific antigens, resulting in efficientantitumor priming of T cells. (Sotomayor E. M. et al., Nature Medicine5:780-787 (1999); Diehl L. et al., Nature Medicine 5:774-9 (1999).)Antitumor activity in the absence of immune cells was also reported byCD40L and anti-CD40 antibody treatment in a human breast cancer linemodel in SCID mice. (Hirano A. et al., Blood 93:2999-3007 (1999).) CD40activation by anti-CD40 antibody was recently shown to eradicate CD40+and CD40− lymphoma in mouse models. (French R. R. et al., NatureMedicine 5:548-53 (1999).) Furthermore, previous studies by Glennie andco-workers conclude that signaling activity by anti-CD40 antibodies ismore effective for inducing in vivo tumor clearance than otheranti-surface marker antibodies capable of recruiting effectors. (Tutt A.L. et al., J. of Immunol. 161:3176-85 (1998).) Consistent with theseobservations, when anti-CD40 antibodies were tested for activity againstCD40+ tumor cells in vivo, most but not all of the tumoricidal activitywas associated with CD40 signaling rather than ADCC. (Funakoshi S. etal., J. of Immunotherapy with Emphasis on Tumor Immunol. 19:93-101(1996).) In another study, bone marrow dendritic cells were treated exvivo with a variety of agents, and tested for in vivo antitumoractivity. These studies demonstrated that CD40L stimulated DCs were themost mature and most effective cells that mounting an antitumorresponse.

The essential role of CD40 in antitumor immunity has also beendemonstrated by comparing responses of wild-type and CD40−/− mice totumor vaccines. These studies show that CD40−/− mice are incapable ofachieving the tumor immunity observed in normal mice. (Mackey M. F. etal., Cancer Research 57:2569-74 (1997).) In another study, splenocytesfrom tumor bearing mice were stimulated with tumor cells and treatedwith activating anti-CD40 antibodies ex vivo, and were shown to haveenhanced tumor specific CTL activity. (Donepudi M. et al., CancerImmunol. Immunother. 48:153-164 (1999).) These studies demonstrate thatCD40 occupies a critical position in antitumor immunity, in both CD40positive and negative tumors. Since CD40 is expressed in lymphomas,leukemias, multiple myeloma, a majority of carcinomas of nasopharynx,bladder, ovary, and liver, and some breast and colorectal cancers,activation of CD40 can have a broad range of clinical applications.

Anti-CD40 activating monoclonal antibodies can contribute to tumoreradication via several important mechanisms. Foremost among these isactivation of host dendritic cells for enhanced tumor antigen processingand presentation, as well as enhanced antigen presentation orimmunogenicity of CD40 positive tumor cells themselves, leading toactivation of tumor specific CD4⁺ and CD8⁺ lymphocytes. Additionalantitumor activity can be mediated by other immune-enhancing effects ofCD40 signaling (production of chemokines and cytokines, recruitment andactivation monocytes, and enhanced CTL and NK cytolytic activity), aswell as direct killing of CD40⁺ tumors by induction of apoptosis or bystimulating a humoral response leading to ADCC. Apoptotic and dyingtumor cells can also become an important source of tumor-specificantigens that are processed and presented by CD40 activated APCs.

Accordingly, there is a critical need for therapeutic, clinicallyrelevant anti-CD40 agonist antibodies.

SUMMARY OF THE INVENTION

The present invention provides an isolated antibody or antigen-bindingportion thereof that binds CD40 and acts as a CD40 agonist.

The invention provides a composition comprising the anti-CD40 antibody,or antigen binding portion thereof, and a pharmaceutically acceptablecarrier. The composition may further comprise another component, such asan anti-tumor agent or an imaging agent. Diagnostic and therapeuticmethods are also provided by the invention.

The invention provides an isolated cell line, such as a hybridoma, thatproduces an anti-CD40 antibody or antigen binding portion thereof.

The invention also provides nucleic acid molecules encoding the heavyand/or light chain, or antigen-binding portions thereof, of an anti-CD40antibody.

The invention provides vectors and host cells comprising the nucleicacid molecules, as well as methods of recombinantly producing thepolypeptides encoded by nucleic acid molecules.

Non-human transgenic animals that express the heavy and/or light chain,or antigen-binding portions thereof, of an anti-CD40 antibody are alsoprovided.

The invention also provides a method for treating a subject in needthereof with an effective amount of a nucleic acid molecule encoding theheavy and/or light chain, or antigen-binding portions thereof, of ananti-CD40 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H are sequence alignments of predicted amino acid sequences ofisolated anti-CD40 monoclonal antibody light and heavy chain variabledomains with the germline amino acid sequences of the correspondinglight and heavy chain genes.). Differences between the clones and thegermline sequence are indicated by shading. The germline CDR1, CDR2, andCDR3 sequences are underlined. In alignments of heavy chain sequences,apparent insertions to the CDR3 region are indicated by a dash (−) inthe germline sequence and apparent deletions in the CDR3 region areindicated by a dash (−) in the clone sequence.

FIG. 1A: the predicted kappa light chain variable region amino acidsequences of mAbs 3.1.1 (SEQ ID NO: 4) and 7.1.2 (SEQ ID NO: 12) withthe Vκ-=A3/A19 and Jκ1 gene germline (SEQ ID NO: 103) amino acidsequences.

FIG. 1B: the predicted kappa light chain variable region amino acidsequence from clone 15.1.1 (SEQ ID NO: 28) and the germline amino acidsequence (Vκ=A3/A19 and J=Jκ2) (SEQ ID NO: 104);

FIG. 1C: the predicted kappa light chain variable region amino acidsequences from mAbs 10.8.3 (SEQ ID NO: 20) and 21.4.1 (SEQ ID NO: 44)and the germline amino acid sequence (Vκ=L5 (DP5) and J=Jκ4) (SEQ ID NO:105);

FIG. 1D: the predicted heavy chain variable region amino acid sequencefrom mAb 3.1.1 (SEQ ID NO: 2) and the germline amino acid sequence(V_(H)=3-30+(DP-49), D=D4+DIR3 and J=J_(H)6) (SEQ ID NO: 106);

FIG. 1E: the predicted heavy chain variable region amino acid sequencefrom mAb 7.1.2 (SEQ ID NO: 10) and the germline amino acid sequence(V_(H)=3-30+ (DP-49), D=DIR5+D1-26 and J=J_(H)6) (SEQ ID NO: 107);

FIG. 1F: the predicted heavy chain amino acid sequences from mAb 10.8.3(SEQ ID NO: 18): and the germline amino acid sequence (V_(H)=4.35(VIV-4), D=DIR3 and J=J_(H)6) (SEQ ID NO: 108);

FIG. 1G: the predicted heavy chain variable region amino acid sequencesfrom mAb 15.1.1 (SEQ ID NO: 26) and the germline amino acid sequence(V_(H)=4-59 (DP-71), D=D4-23 and J=J_(H)4) (SEQ ID NO: 109); and

FIG. 1H: the predicted heavy variable region chain amino acid sequencesfrom mAb 21.4.1 (SEQ ID NO: 42) and the germline amino acid sequence(V_(H)=1-02 (DP-75), D=DLR1 and J=J_(H)4) (SEQ ID NO: 110).

FIGS. 2A-2H are sequence alignments of predicted amino acid sequences ofisolated anti-CD40 monoclonal antibody light and heavy chain variabledomains with the germline amino acid sequences of the correspondinglight and heavy chain genes.). Differences between the clones and thegermline sequence are indicated in bold. The germline CDR1, CDR2, andCDR3 sequences are underlined. In alignments of heavy chain sequences,apparent insertions to the CDR3 region are indicated by a dash (−) inthe germline sequence and apparent deletions in the CDR3 region areindicated by a dash (−) in the clone sequence.

FIG. 2A: the predicted kappa light chain amino acid sequences from mabs22.1.1 (SEQ ID NO: 52), 23.5.1 (SEQ ID NO: 60) and 23.29.1 (SEQ ID NO:76) and the germline amino acid sequence (Vκ=A3/A19 and J=Jκ1) (SEQ IDNO: 111);

FIG. 2B: the predicted kappa light chain amino acid sequence from mAb21.2.1 (SEQ ID NO: 36) and the germline amino acid sequence (Vκ=A3/A19and J=Jκ3) (SEQ ID NO: 112);

FIG. 2C: the predicted kappa light chain amino acid sequences from mAbs23.28.1 (SEQ ID NO: 68), 23.28.1L-C92A (SEQ ID NO: 100) and 24.2.1 (SEQID NO: 84) and the germline amino acid sequence (Vκ=A27 and J=Jκ3); (SEQID NO: 113);

FIG. 2D: the predicted heavy chain amino acid sequence from mAb 21.2.1(SEQ ID NO: 34) and the germline amino acid sequence (V_(H)=3-30+,D=DIR3+D6-19 and J=J_(H)4) (SEQ ID NO: 114);

FIG. 2E: the predicted heavy chain amino acid sequence from mAbs 22.1.1(SEQ ID NO: 50), 22.1.1H-C109A (SEQ ID NO: 96) and the germline aminoacid sequence (V_(H)=3-30-30+, D=D1-1 and J=J_(H)6) (SEQ ID NO: 115);

FIG. 2F: the predicted heavy chain amino acid sequence from mAb 23.5.1(SEQ ID NO: 58) and the germline amino acid sequence (V_(H)=3-30+,D=D4-17 and J=J_(H)6) (SEQ ID NO: 116);

FIG. 2G: the predicted heavy chain amino acid sequence from mAb 23.29.1(SEQ ID NO: 74) and the germline amino acid sequence (V_(H)=3-30.3,D=D4-17 and J=J_(H)6) (SEQ ID NO: 117); and

FIG. 2H: the predicted heavy chain amino acid sequences from mAb 23.28.1(SEQ ID NO: 66), 23.28.1H-D16E (SEQ ID NO: 98) and 24.2.1 (SEQ ID 30 NO:82) and the germline amino acid sequence (V_(H)=4-59, D=DIR1+D4-17 andJ=J_(H)5) (SEQ ID NO: 142).

FIG. 3 is a dose-response curve that illustrates the ability of ananti-CD40 antibody of the invention (21.4.1) to enhance IL-12p40production by human dendritic cells.

FIG. 4 is a dose-response curve that illustrates the ability of ananti-CD40 antibody of the invention (21.4.1) to enhance IL-12p70production by human dendritic cells.

FIG. 5 is a graph that illustrates the ability of an anti-CD40 antibodyof the invention (21.4.1) to increase immunogenicity of Jy stimulatorcells and enhance CTL activity against Jy target cells.

FIG. 6 is a tumor growth inhibition curve that illustrates the reducedgrowth of CD40 positive Daudi tumors in SCID-beige mice treated with ananti-CD40 antibody of the invention (21.4.1).

FIG. 7 is a tumor growth inhibition curve that illustrates the reducedgrowth of CD40 negative K562 tumors in SCID-beige mice treated with ananti-CD40 antibody of the invention (21.4.1) and human dendritic cellsand T cells.

FIG. 8 shows inhibition in the growth of CD40 negative K562 tumors inSCID mice by different concentrations of anti-CD40 agonist mAb 23.29.1.

FIG. 9 shows inhibition in the growth of CD40 negative K562 tumors inSCID mice by different concentrations of anti-CD40 agonist mAb 3.1.1.

FIG. 10 shows inhibition in the growth of CD40 positive Raji tumors inthe presence and absence of T cells and dendritic cells in SCID mice byan anti-CD40 agonist mAb.

FIG. 11 shows inhibition in the growth of CD40 positive Raji tumors inSCID mice by anti-CD40 agonist antibodies.

FIG. 12 shows inhibition in the growth of BT 474 breast cancer cells inSCID-beige mice by anti-CD40 agonist antibodies.

FIG. 13 shows inhibition in the growth of PC-3 prostate tumors inSCID-beige mice by anti-CD40 agonist antibodies.

FIG. 14 is a survival curve for SCID-beige mice injected (iv) with Dauditumor cells and treated with anti-CD40 agonist antibodies.

FIG. 15 is a Western blot analysis of anti-CD40 agonist antibodies toreduced (R) and non-reduced (NR) human CD40.

FIG. 16 is an alignment of the D1-D4 domains of mouse and human CD40.

FIG. 17 is an alignment of the mouse and human CD40 amino acid sequencesshowing the location of the fusion sites of the chimeras.

FIG. 18 is a group of schematic diagrams of the chimeric CD40constructs.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and General Techniques

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well known and commonly used in the art.

The methods and techniques of the present invention are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992), and Harlow and Lane Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990), which are incorporated herein by reference.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclatures used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “isolated protein”, “isolated polypeptide” or “isolatedantibody” is a protein, polypeptide or antibody that by virtue of itsorigin or source of derivation (1) is not associated with naturallyassociated components that accompany it in its native state, (2) is freeof other proteins from the same species, (3) is expressed by a cell froma different species, or (4) does not occur in nature. Thus, apolypeptide that is chemically synthesized or synthesized in a cellularsystem different from the cell from which it naturally originates willbe “isolated” from its naturally associated components. A protein mayalso be rendered substantially free of naturally associated componentsby isolation, using protein purification techniques well known in theart.

Examples of isolated antibodies include an anti-CD40 antibody that hasbeen affinity purified using CD40, an anti-CD40 antibody that has beensynthesized by a hybridoma or other cell line in vitro, and a humananti-CD40 antibody derived from a transgenic mouse.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous,” or “substantially purified” when at least about 60 to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/Wof a protein sample, more usually about 95%, and preferably will be over99% pure. Protein purity or homogeneity may be indicated by a number ofmeans well known in the art, such as polyacrylamide gel electrophoresisof a protein sample, followed by visualizing a single polypeptide bandupon staining the gel with a stain well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence. In some embodiments,fragments are at least 5, 6, 8 or 10 amino acids long. In otherembodiments, the fragments are at least 14, at least 20, at least 50, orat least 70, 80, 90, 100, 150 or 200 amino acids long.

The term “polypeptide analog” as used herein refers to a polypeptidethat comprises a segment that has substantial identity to a portion ofan amino acid sequence and that has at least one of the followingproperties: (1) specific binding to CD40 under suitable bindingconditions, (2) ability to activate CD40, (3) the ability to upregulatethe expression of cell surface molecules such as ICAM, MHC-II, B7-1,B7-2, CD71, CD23 and CD83, or (4) the ability to enhance the secretionof cytokines such as IFN-β1, IL-2, IL-8, IL-12, IL-15, IL-18 and IL-23.Typically, polypeptide analogs comprise a conservative amino acidsubstitution (or insertion or deletion) with respect to thenaturally-occurring sequence. Analogs typically are at least 20 or 25amino acids long, preferably at least 50, 60, 70, 80, 90, 100, 150 or200 amino acids long or longer, and can often be as long as afull-length naturally-occurring polypeptide.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, and (4) conferor modify other physicochemical or functional properties of suchanalogs. Analogs can include various muteins of a sequence other thanthe naturally-occurring peptide sequence. For example, single ormultiple amino acid substitutions (preferably conservative amino acidsubstitutions) may be made in the naturally-occurring sequence(preferably in the portion of the polypeptide outside the domain(s)forming intermolecular contacts). A conservative amino acid substitutionshould not substantially change the structural characteristics of theparent sequence (e.g., a replacement amino acid should not tend to breaka helix that occurs in the parent sequence, or disrupt other types ofsecondary structure that characterizes the parent sequence). Examples ofart-recognized polypeptide secondary and tertiary structures aredescribed in Proteins, Structures and Molecular Principles (Creighton,Ed., W. H. Freeman and Company, New York (1984)); Introduction toProtein Structure (C. Branden and J. Tooze, eds., Garland Publishing,New York, N.Y. (1991)); and Thornton et al., Nature 354:105 (1991),which are each incorporated herein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry asdrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics.” Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber andFreidinger, TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30:1229(1987), which are incorporated herein by reference. Such compounds areoften developed with the aid of computerized molecular modeling. Peptidemimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent therapeutic orprophylactic effect. Generally, peptidomimetics are structurally similarto a paradigm polypeptide (i.e., a polypeptide that has a desiredbiochemical property or pharmacological activity), such as a humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of: —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—,by methods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may also be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch, Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

An “antibody” refers to a complete antibody or to an antigen-bindingportion thereof, that competes with the intact antibody for specificbinding. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed.,2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in itsentirety for all purposes). Antigen-binding portions may be produced byrecombinant DNA techniques or by enzymatic or chemical cleavage ofintact antibodies. Antigen-binding portions include, inter alia, Fab,Fab′, F(ab′)₂, Fd, Fv, dAb, and complementarity determining region (CDR)fragments, single-chain antibodies (scFv), chimeric antibodies,diabodies and polypeptides that contain at least a portion of anantibody that is sufficient to confer specific antigen binding to thepolypeptide.

From N-terminus to C-terminus, both light and heavy chain variabledomains comprise the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.The assignment of amino acids to each domain is in accordance with thedefinitions of Kabat, Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature342:878-883 (1989).

As used herein, an antibody that is referred to by number is amonoclonal antibody that is obtained from the hybridoma of the samenumber. For example, monoclonal antibody 3.1.1 is obtained fromhybridoma 3.1.1.

As used herein, a Fd fragment means an antibody fragment that consistsof the V_(H) and C_(H) 1 domains; an Fv fragment consists of the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment(Ward et al., Nature 341:544-546 (1989)) consists of a V_(H) domain.

In some embodiments, the antibody is a single-chain antibody (scFv) inwhich a V_(L) and V_(H) domains are paired to form a monovalentmolecules via a synthetic linker that enables them to be made as asingle protein chain. (Bird et al., Science 242:423-426 (1988) andHuston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988).) In someembodiments, the antibodies are diabodies, i.e., are bivalent antibodiesin which V_(H) and V_(L) domains are expressed on a single polypeptidechain, but using a linker that is too short to allow for pairing betweenthe two domains on the same chain, thereby forcing the domains to pairwith complementary domains of another chain and creating two antigenbinding sites. (See e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA90:6444-6448 (1993), and Poljak R. J. et al., Structure 2:1121-1123(1994).) In some embodiments, one or more CDRs from an antibody of theinvention may be incorporated into a molecule either covalently ornoncovalently to make it an immunoadhesin that specifically binds toCD40. In such embodiments, the CDR(s) may be incorporated as part of alarger polypeptide chain, may be covalently linked to anotherpolypeptide chain, or may be incorporated noncovalently.

In embodiments having one or more binding sites, the binding sites maybe identical to one another or may be different.

As used herein, the term “human antibody” means any antibody in whichall of the variable and constant domain sequences are human sequences.These antibodies may be prepared in a variety of ways, as describedbelow.

The term “chimeric antibody” as used herein means an antibody thatcomprises regions from two or more different antibodies. In oneembodiment, one or more of the CDRs are derived from a human anti-CD40antibody. In another embodiment, all of the CDRs are derived from ahuman anti-CD40 antibody. In another embodiment, the CDRs from more thanone human anti-CD40 antibodies are combined in a chimeric antibody. Forinstance, a chimeric antibody may comprise a CDR1 from the light chainof a first human anti-CD40 antibody, a CDR2 from the light chain of asecond human anti-CD40 antibody and a CDR3 and CDR3 from the light chainof a third human anti-CD40 antibody, and the CDRs from the heavy chainmay be derived from one or more other anti-CD40 antibodies. Further, theframework regions may be derived from one of the same anti-CD40antibodies or from one or more different human.

An “activating antibody” (also referred to herein as an “agonistantibody” as used herein means an antibody that increases one or moreCD40 activities by at least about 20% when added to a cell, tissue ororganism expressing CD40. In some embodiments, the antibody activatesCD40 activity by at least 40%, 50%, 60%, 70%, 80%, 85%. In someembodiments, the activating antibody is added in the presence of CD40L.In some embodiments, the activity of the activating antibody is measuredusing a whole blood surface molecule upregulation assay. See ExampleVII. In another embodiment, the activity of the activating antibody ismeasured using a dendritic cell assay to measure IL-12 release. SeeExample VIII. In another embodiment the activity of the activatingantibody is measured using an in vivo tumor model. See Example X.

Fragments or analogs of antibodies or immunoglobulin molecules can bereadily prepared by those of ordinary skill in the art following theteachings of this specification. Preferred amino- and carboxy-termini offragments or analogs occur near boundaries of functional domains.Structural and functional domains can be identified by comparison of thenucleotide and/or amino acid sequence data to public or proprietarysequence databases. Preferably, computerized comparison methods are usedto identify sequence motifs or predicted protein conformation domainsthat occur in other proteins of known structure and/or function. Methodsto identify protein sequences that fold into a known three-dimensionalstructure are known. See Bowie et al., Science 253:164 (1991).

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore™ system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson U. et al., Ann. Biol. Clin. 51:19-26(1993); Jonsson U. et al., Biotechniques 11:620-627 (1991); Jonsson B.et al., J. Mol. Recognit. 8:125-131 (1995); and Johnsson B. et al.,Anal. Biochem. 198:268-277 (1991).

The term “K_(D)” refers to the equilibrium dissociation constant of aparticular antibody-antigen interaction.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. An antibody is said to specifically bind an antigenwhen the equilibrium dissociation constant is ≦1 μM, preferably ≦100 nMand most preferably ≦10 nM.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms.

The term “isolated polynucleotide” as used herein means a polynucleotideof genomic, cDNA, or synthetic origin or some combination thereof, whichby virtue of its origin the “isolated polynucleotide” (1) is notassociated with all or a portion of a polynucleotides with which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide to which it is not linked in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “oligonucleotide” as used herein includes naturally occurring,and modified nucleotides linked together by naturally occurring andnon-naturally occurring oligonucleotide linkages. Oligonucleotides are apolynucleotide subset generally comprising a length of 200 bases orfewer. Preferably oligonucleotides are 10 to 60 bases in length and mostpreferably 12, 13, 14, 15, 16, 17, 18, 19, or to 40 bases in length.Oligonucleotides are usually single stranded, e.g. for primers andprobes; although oligonucleotides may be double stranded, e.g. for usein the construction of a gene mutant. Oligonucleotides of the inventioncan be either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” as used herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” as used herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al., Nucl. AcidsRes. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984);Stein et al., Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti-CancerDrug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); U.S. Pat. No. 5,151,510; Uhlmann andPeyman, Chemical Reviews 90:543 (1990), the disclosures of which arehereby incorporated by reference. An oligonucleotide can include a labelfor detection, if desired.

“Operably linked” sequences include both expression control sequencesthat are contiguous with the gene of interest and expression controlsequences that act in trans or at a distance to control the gene ofinterest. The term “expression control sequence” as used herein meanspolynucleotide sequences that are necessary to effect the expression andprocessing of coding sequences to which they are ligated. Expressioncontrol sequences include appropriate transcription initiation,termination, promoter and enhancer sequences; efficient RNA processingsignals such as splicing and polyadenylation signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance proteinsecretion. The nature of such control sequences differs depending uponthe host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

The term “vector”, as used herein, means a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. Insome embodiments, the vector is a plasmid, i.e., a circular doublestranded DNA loop into which additional DNA segments may be ligated. Insome embodiments, the vector is a viral vector, wherein additional DNAsegments may be ligated into the viral genome. In some embodiments, thevectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors). In other embodiments,the vectors (e.g., non-episomal mammalian vectors) can be integratedinto the genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome. Moreover, certainvectors are capable of directing the expression of genes to which theyare operatively linked. Such vectors are referred to herein as“recombinant expression vectors” (or simply, “expression vectors”).

The term “recombinant host cell” (or simply “host cell”), as usedherein, means a cell into which a recombinant expression vector has beenintroduced. It should be understood that “recombinant host cell” and“host cell” mean not only the particular subject cell but also theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. “High stringency” or “highly stringent” conditions can beused to achieve selective hybridization conditions as known in the artand discussed herein. One example of “high stringency” or “highlystringent” conditions is the incubation of a polynucleotide with anotherpolynucleotide, wherein one polynucleotide may be affixed to a solidsurface such as a membrane, in a hybridization buffer of 6×SSPE or SSC,50% formamide, 5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured,fragmented salmon sperm DNA at a hybridization temperature of 42° C. for12-16 hours, followed by twice washing at 55° C. using a wash buffer of1×SSC, 0.5% SDS. See also Sambrook et al., supra, pp. 9.50-9.55.

The term “percent sequence identity” in the context of nucleic acidsequences means the residues in two sequences that are the same whenaligned for maximum correspondence. The length of sequence identitycomparison may be over a stretch of at least about nine nucleotides,usually at least about 18 nucleotides, more usually at least about 24nucleotides, typically at least about 28 nucleotides, more typically atleast about 32 nucleotides, and preferably at least about 36, 48 or morenucleotides. There are a number of different algorithms known in the artwhich can be used to measure nucleotide sequence identity. For instance,polynucleotide sequences can be compared using FASTA, Gap or Bestfit,which are programs in Wisconsin Package Version 10.0, Genetics ComputerGroup (GCG), Madison, Wis. FASTA, which includes, e.g., the programsFASTA2 and FASTA3, provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996);Pearson, J. Mol. Biol. 276:71-84 (1998); herein incorporated byreference). Unless otherwise specified, default parameters for aparticular program or algorithm are used. For instance, percent sequenceidentity between nucleic acid sequences can be determined using FASTAwith its default parameters (a word size of 6 and the NOPAM factor forthe scoring matrix) or using Gap with its default parameters as providedin GCG Version 6.1, herein incorporated by reference.

A reference to a nucleotide sequence encompasses its complement unlessotherwise specified. Thus, a reference to a nucleic acid having aparticular sequence should be understood to encompass its complementarystrand, with its complementary sequence.

In the molecular biology art, researchers use the terms “percentsequence identity”, “percent sequence similarity” and “percent sequencehomology” interchangeably. In this application, these terms shall havethe same meaning with respect to nucleic acid sequences only.

The term “substantial similarity” or “substantial sequence similarity,”when referring to a nucleic acid or fragment thereof, means that whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 85%, preferably at leastabout 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99%of the nucleotide bases, as measured by any well-known algorithm ofsequence identity, such as FASTA, BLAST or Gap, as discussed above.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 70, 75 or 80percent sequence identity, preferably at least 90 or 95 percent sequenceidentity, and more preferably at least 97, 98 or 99 percent sequenceidentity. Preferably, residue positions that are not identical differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of similarity may be adjustedupwards to correct for the conservative nature of the substitution.Means for making this adjustment are well-known to those of skill in theart. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 (1994). Examplesof groups of amino acids that have side chains with similar chemicalproperties include 1) aliphatic side chains: glycine, alanine, valine,leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine andthreonine; 3) amide-containing side chains: asparagine and glutamine; 4)aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basicside chains: lysine, arginine, and histidine; 6) acidic side chains:aspartic acid and glutamic acid; and 7) sulfur-containing side chains:cysteine and methionine. Preferred conservative amino acids substitutiongroups are: valine-leucine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, glutamate-aspartate, andasparagine-glutamine.

Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al., Science 256:1443-45 (1992), herein incorporated by reference. A“moderately conservative” replacement is any change having a nonnegativevalue in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “Bestfit” whichcan be used with default parameters to determine sequence homology orsequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000)). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially blastp or tblastn, using default parameters. See, e.g.,Altschul et al., J. Mol. Biol. 215:403-410 (1990); Altschul et al.,Nucleic Acids Res. 25:3389-402 (1997); herein incorporated by reference.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.

As used herein, the terms “label” or “labeled” refers to incorporationof another molecule in the antibody. In one embodiment, the label is adetectable marker, e.g., incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (e.g., streptavidin containing a fluorescent marker orenzymatic activity that can be detected by optical or colorimetricmethods). In another embodiment, the label or marker can be therapeutic,e.g., a drug conjugate or toxin. Various methods of labelingpolypeptides and glycoproteins are known in the art and may be used.Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthamide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (e.g., leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags),magnetic agents, such as gadolinium chelates, toxins such as pertussistoxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. In some embodiments, labels are attached by spacerarms of various lengths to reduce potential steric hindrance.

The term patient includes human and veterinary subjects.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Human Anti-CD40 Antibodies and Characterization Thereof

Human antibodies avoid certain of the problems associated withantibodies that possess non-human (e.g., rodent) variable and/orconstant regions. Such problems include the rapid clearance of theantibodies or immune response against the antibody. Therefore, in oneembodiment, the invention provides humanized anti-CD40 antibodies. Inanother embodiment, the invention provides human anti-CD40 antibodies.In some embodiments, human anti-CD40 antibodies are produced byimmunizing a rodent whose genome comprises human immunoglobulin genes sothat the rodent produces human antibodies. Human anti-CD40 antibodiesare expected to minimize the immunogenic and allergic responsesintrinsic to non-human or non-human-derivatized monoclonal antibodies(Mabs) and thus to increase the efficacy and safety of the administeredantibodies. The use of fully human antibodies can be expected to providea substantial advantage in the treatment of chronic and recurring humandiseases, such as inflammation and cancer, which may require repeatedantibody administrations.

The invention provides eleven activating human anti-CD40 monoclonalantibodies (mAbs) and the hybridoma cell lines that produce them. TableA lists the sequence identifiers (SEQ ID NOS:) of the nucleic acidsencoding the full-length heavy and light chains (including leadersequence), the corresponding full-length deduced amino acid sequences,and the nucleotide and deduced amino acid sequence of the heavy andlight chain variable regions.

TABLE A HUMAN ANTI-CD40 ANTIBODIES SEQUENCE IDENTIFIER (SEQ ID NO:)Variable Region Full Length Heavy Light Heavy Light MAb DNA Protein DNAProtein DNA Protein DNA Protein 3.1.1 1 2 3 4 5 6 7 8 7.1.2 9 10 11 1213 14 15 16 10.8.3 17 18 19 20 21 22 23 24 15.1.1 25 26 27 28 29 30 3132 21.2.1 33 34 35 36 37 38 39 40 21.4.1 41 42 43 44 45 46 47 48 22.1.149 50 51 52 53 54 55 56 23.5.1 57 58 59 60 61 62 63 64 23.28.1 65 66 6768 69 70 71 72 23.29.1 73 74 75 76 77 78 79 80 24.2.1 81 82 83 84 85 8687 88

The invention further provides human anti-CD40 mAb 23.25.1 and thehybridoma cell line that produces it.

The invention further provides heavy and/or light chain variants ofcertain of the above-listed human anti-CD40 mAbs, comprising one or moreamino acid substitutions. The invention provides two variant heavychains of mAb 3.1.1. In one, the alanine at residue 78 is changed tothreonine. In the second, the alanine at residue 78 is changed tothreonine, and the valines at residues 88 and 97 are changed toalanines. The invention also provides a variant light chain of mAb 3.1.1in which the leucine at residue 4 and the leucine at residue 83 arechanged to methionine and valine, respectively. Combination with avariant heavy or light chain with a wild type light or heavy chain,respectively is designated by the mutant chain. Thus, an antibodycontaining a wild type light chain and a heavy chain comprising thealanine to threonine mutation at residue 78 is designated as3.1.1H-A78T. However, in other embodiments of the invention, antibodiescontaining any combination of a variant heavy chain and the variantlight chain of 3.1.1 are included.

Further, the invention provides a variant of the heavy chain of mAb22.1.1 in which the cysteine at residue 109 is changed to an alanine. Amonoclonal antibody comprising the variant heavy chain and the 22.1.1light chain is designated mAb 22.1.1H-C109A. The invention furtherprovides two variant heavy chains and a variant light chain of mAb23.28.1. In one heavy chain variant, the aspartic acid at residue 16 ischanged to glutamic acid. A mAb comprising the variant heavy chainvariant and the 23.28.1 light chain is designated 23.28.1H-D16E. Theinvention also includes a 23.28.1 light chain variant in which thecysteine at residue 92 is changed to an alanine. A mAb comprising the23.28.1 heavy chain and the variant light chain is designated 23.28.1 LC92A. The invention also provides mAbs comprising either of the 23.28.1heavy chain variants with the 23.28.1 light chain variant.

The light chain produced by hybridoma 23.29.1 contains a mutation in theconstant region at residue 174. The light chain produced by thehybridoma has arginine at this position instead of the canonical lysine.Accordingly, the invention also provides a 23.29.1 light chain with thecanonical lysine at residue 174 and a mAb, designated 23.29.1L-R174K,comprising the 23.29.1 heavy chain and the variant light chain.

In a preferred embodiment, the anti-CD40 antibody is 3.1.1, 3.1.1H-A78T,3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1,21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1H-D16E,23.28.1L-C92A, 23.29.1, 23.29.1L-R174K and 24.2.1. In some embodiments,the anti-CD40 antibody comprises a light chain comprising an amino acidsequence selected from SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80,88, 94, 100 or 102 or the variable region therefrom, or encoded by anucleic acid sequence selected from SEQ ID NO: 7, 15, 23, 31, 39, 47,55, 63, 71, 79, 87, 93, 99 or 101. In some embodiments, the anti-CD40antibody comprises a light chain comprising at least the CDR2 from oneof listed antibodies, one of the above-identified amino acid sequences(as shown in FIGS. 1A-1C and 2A-2C) or encoded by one of theabove-identified nucleic acid sequences. In another embodiment, thelight chain further comprises a CDR1 and CDR3 independently selectedfrom a light chain variable region that comprises no more than ten aminoacids from the amino acid sequence encoded by a germline Vκ A3/A19, L5or A27 gene, or comprises a CDR1 and CDR3 independently selected fromone of a CDR 1 and CDR3 of (1) an antibody selected from 3.1.1,3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1,23.25.1, 23.28.1, 23.28.1L-C92A, 23.29.1, 23.29.1L-R174K or 24.2.1; (2)the amino acid sequence of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68,76, 84, 94, 100 or 102 or (3) encoded by the nucleic acid sequence ofSEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 93, 99 or 101.

In another preferred embodiment, the anti-CD40 antibody comprises aheavy chain comprising an amino acid sequence selected from SEQ ID NOS:6, 14, 22, 30, 38, 46, 54, 62, 70, 78 or 86 or the variable regiontherefrom or encoded by a nucleic acid sequence selected from SEQ IDNOS: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77 or 85. In some embodiments,the anti-CD40 antibody comprises a heavy chain comprising at least theCDR3 from one of listed antibodies, one of the above-identified aminoacid sequences (as shown in FIGS. 1A-1C and 2A-2C) or encoded by one ofthe above-identified nucleic acid sequences. In another embodiment, theheavy chain further comprises a CDR1 and CDR2 independently selectedfrom a heavy chain variable region that comprises no more than eighteenamino acids from the amino acid sequence encoded by a germline V_(H)3-30+, 4-59, 1-02, 4.35 or 3-30.3 gene, or comprises a CDR1 and CDR2independently selected from one of a CDR1 and CDR2 of(1) an antibodyselected from 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 7.1.2, 10.8.3,15.1.1, 21.4.1, 21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1,23.28.1H-D16E, 23.29.1 and 24.2.1; (2) the amino acid sequence of SEQ IDNO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 92, 96 or 98 or (3)encoded by the nucleic acid sequence of SEQ ID NO: 1, 9, 17, 25, 33, 41,49, 57, 65, 73, 81, 89, 91, 95 or 97. In another embodiment, theanti-CD40 antibody comprises a heavy chain and a light chain as definedabove.

As used herein, antibody 3.1.1H-A78T is identical to that of 3.1.1except that residue 78 of the heavy chain is threonine instead ofalanine. Similarly, in antibody 3.1.1H-A78T-V88A-V97A, residue 78 ischanged to A, and residues 88 and 97 are changed from valine to alaninein the heavy chain. Antibody 3.1.1 L-L4M-L83V is identical to that of3.1.1 except that residue 4 is methionine instead of leucine and residue83 is valine instead of leucine in the light chain. Antibody22.1.1H-C109A is identical to that of 22.1.1 except that residue 109 ofthe heavy chain is changed from a cysteine to an alanine. Antibodies23.28.1H-D16E and 23.28.1 L-C92A are identical to that of 23.28.1 exceptthat residue 16 of the heavy chain is changed from aspartate toglutamate, and residue 92 of the light chain is changed from cysteine toalanine, respectively. Antibody 23.29.1L-R174K is identical to that of23.29.1 except that residue 174 of the light chain is changed fromarginine to lysine.

Class and Subclass of Anti-CD40 Antibodies

The class and subclass of anti-CD40 antibodies may be determined by anymethod known in the art. In general, the class and subclass of anantibody may be determined using antibodies that are specific for aparticular class and subclass of antibody. Such antibodies are availablecommercially. The class and subclass can be determined by ELISA, orWestern Blot as well as other techniques. Alternatively, the class andsubclass may be determined by sequencing all or a portion of theconstant domains of the heavy and/or light chains of the antibodies,comparing their amino acid sequences to the known amino acid sequencesof various class and subclasses of immunoglobulins, and determining theclass and subclass of the antibodies.

In some embodiments, the anti-CD40 antibody is a monoclonal antibody.The anti-CD40 antibody can be an IgG, an IgM, an IgE, an IgA or an IgDmolecule. In a preferred embodiment, the anti-CD40 antibody is an IgGand is an IgG1, IgG2, IgG3 or IgG4 subclass. In another preferredembodiment, the anti-CD40 antibodies are subclass IgG2.

Species and Molecule Selectivity

In another aspect of the invention, the anti-CD40 antibodies demonstrateboth species and molecule selectivity. In some embodiments, theanti-CD40 antibody binds to primate and human CD40. In some embodiments,the anti-CD40 antibody binds to human, cynomolgus or rhesus CD40. Inother embodiments, the anti-CD40 antibody does not bind to mouse, rat,dog or rabbit CD40. Following the teachings of the specification, onecan determine the species selectivity for the anti-CD40 antibody usingmethods well known in the art. For instance, one can determine speciesselectivity using Western blot, FACS, ELISA or RIA. (See, e.g., ExampleIV.)

In some embodiments, the anti-CD40 antibody has a selectivity for CD40that is more than 100 times greater than its selectivity for RANK(receptor activator of nuclear factor-kappa B), 4-1BB (CD137), TNFR-1(Tumor Necrosis Factor Receptor-1) and TNFR-2 (Tumor Necrosis FactorReceptor-2). In some embodiments, the anti-CD40 antibody does notexhibit any appreciable specific binding to any other protein other thanCD40. One can determine the selectivity of the anti-CD40 antibody forCD40 using methods well known in the art following the teachings of thespecification. For instance, one can determine the selectivity usingWestern blot, FACS, ELISA or RIA. (See, e.g., Example V.)

Identification of CD40 Epitopes Recognized by Anti-CD40 Antibody

Further, the invention provides a human anti-CD40 monoclonal antibodythat binds CD40 and cross-competes with and/or binds the same epitopeand/or binds to CD40 with the same K_(D) as a human anti-CD40 antibodyselected from an antibody 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A,3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1,22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1H-D16E, 23.28.1L-C92A,23.29.1, 23.29.1L-R174K or 24.2.1; or a human anti-CD40 antibody thatcomprises a heavy chain variable region having an amino acid sequence ofSEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 92, 96 or 98or a human anti-CD40 antibody that comprises a light chain variableregion having an amino acid sequence of SEQ ID NO: 4, 12, 20, 28, 36,44, 52, 60, 68, 76, 84, 94, 100 or 102.

One can determine whether an antibody binds to the same epitope as orcross competes for binding with an anti-CD40 antibody by using anymethod known in the art. In one embodiment, one can allow the anti-CD40antibody of the invention to bind to CD40 under saturating conditionsand then measure the ability of the test antibody to bind to CD40. Ifthe test antibody is able to bind to the CD40 at the same time as theanti-CD40 antibody, then the test antibody binds to a different epitopeas the anti-CD40 antibody. However, if the test antibody is not able tobind to the CD40 at the same time, then the test antibody binds to thesame epitope, an overlapping epitope, or an epitope that is in closeproximity to the epitope bound by the human anti-CD40 antibody. Thisexperiment can be performed using ELISA, RIA, FACS or surface plasmonresonance. (See, e.g., Example VI.) In a preferred embodiment, theexperiment is performed using surface plasmon resonance. In a morepreferred embodiment, BIAcore is used.

Binding Affinity of Anti-CD40 Antibodies to CD40

In some embodiments of the invention, the anti-CD40 antibody binds toCD40 with high affinity. In some embodiments, the anti-CD40 antibodybinds to CD40 with a K_(D) of 2×10⁻⁸ M or less. In another preferredembodiments, the antibody binds to CD40 with a K_(D) of 2×10⁻⁹, 2×10⁻¹⁰,4.0×10⁻¹¹ M or less. In an even more preferred embodiment, the antibodybinds to CD40 with a K_(D) of 2.5×10⁻¹² M or less. In some embodiments,the antibody binds to CD40 with substantially the same K_(D) as anantibody selected from 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A,3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1,22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1H-D16E, 23.28.1L-C92A,23.29.1, 23.29.1L-R174K or 24.2.1. In another preferred embodiment, theantibody binds to CD40 with substantially the same K_(D) as an antibodythat comprises a CDR2 of a light chain, and/or a CDR3 of a heavy chainfrom an antibody selected from 3.1.1, 3.1.1H-A78T,3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1,21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1H-D16E,23.28.1L-C92A, 23.29.1, 23.29.1L-R174K and 24.2.1. In still anotherpreferred embodiment, the antibody binds to CD40 with substantially thesame K_(D) as an antibody that comprises a heavy chain variable regionhaving an amino acid sequence of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50,58, 66, 74, 82, 90, 92, 96 or 98 or that comprises a light chain havingan amino acid sequence of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68,76, 84, 94, 100 or 102. In another preferred embodiment, the antibodybinds to CD40 with substantially the same K_(D) as an antibody thatcomprises a CDR2 of a light chain variable region having an amino acidsequence of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 94,100 or 102 or a CDR3 of a heavy chain variable region having an aminoacid sequence of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82,90, 92, 96 or 98.

In some embodiments, the anti-CD40 antibody has a low dissociation rate.In some embodiments, the anti-CD40 antibody has an K_(off) of 2.0×10⁻⁴or lower. In some embodiments, the k_(off) is 2.0×10⁻⁷ or lower. In someembodiments, the K_(off) is substantially the same as an antibodydescribed herein, including an antibody selected from 3.1.1,3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3,15.1.1, 21.4.1, 21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1,23.28.1H-D16E, 23.28.1L-C92A, 23.29.1, 23.29.1L-R174K and 24.2.1. Insome embodiments, the antibody binds to CD40 with substantially the sameK_(off) as an antibody that comprises a CDR3 of a heavy chain or a CDR2of a light chain from an antibody selected from 3.1.1, 3.1.1H-A78T,3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1,21.2.1, 22.1.1, 22.1.H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1H-D16E,23.28.1L-C92A, 23.29.1, 23.29.1L-R174K and 24.2.1. In some embodiments,the antibody binds to CD40 with substantially the same K_(off) as anantibody that comprises a heavy chain variable region having an aminoacid sequence of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82,90, 92, 96 or 98 or that comprises a light chain variable region havingan amino acid sequence of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68,76, 84, 94, 100 or 102. In another preferred embodiment, the antibodybinds to CD40 with substantially the same K_(off) as an antibody thatcomprises a CDR2 of a light chain variable region having an amino acidsequence of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 94,100 or 102 or a CDR3 of a heavy chain variable region having an aminoacid sequence of SEQ ID NO: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86,90, 92, 96 or 98.

The binding affinity and dissociation rate of an anti-CD40 antibody toCD40 can be determined by any method known in the art. The bindingaffinity can be measured by competitive ELISAs, RIAs or surface plasmonresonance, such as BIAcore. The dissociation rate also can be measuredby surface plasmon resonance. Preferably, the binding affinity anddissociation rate is measured by surface plasmon resonance. Morepreferably, the binding affinity and dissociation rate are measuredusing a BIAcore™. See, e.g., Example XIV.

Light and Heavy Chain Gene Usage

An anti-CD40 antibody of the invention can comprise a human kappa or ahuman lambda light chain or an amino acid sequence derived therefrom. Insome embodiments comprising a kappa light chain, the light chainvariable domain (V_(L)) is encoded in part by a human A3/A19 (DPK-15),L5 (DP5), or A27 (DPK-22) Vκ gene.

In some embodiments, the V_(L) of the anti-CD40 antibody contains one ormore amino acid substitutions relative to the germline amino acidsequence. In some embodiments, the V_(L) of the anti-CD40 antibodycomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutionsrelative to the germline amino acid sequence. In some embodiments, oneor more of those substitutions from germline is in the CDR regions ofthe light chain. In some embodiments, the amino acid substitutionsrelative to germline are at one or more of the same positions as thesubstitutions relative to germline in any one or more of the V_(L) ofantibodies 3.1.1, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1,21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1, 23.28.1L-C92A, 23.29.1,23.29.1L-R174K and 24.2.1. For example, the V_(L) of the anti-CD40antibody may contain one or more amino acid substitutions compared togermline found in antibody 21.4.1, and other amino acid substitutionscompared to germine found in antibody 10.8.3 which utilizes the same Vκgene as antibody 21.4.1. In some embodiments, the amino acid changes areat one or more of the same positions but involve a different mutationthan in the reference antibody.

In some embodiments, amino acid changes relative to germline occur atone or more of the same positions as in any of the V_(L) of antibodies3.1.1, 3.1.1 L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1,23.5.1, 23.25.1, 23.28.1, 23.28.1L-C92A, 23.29.1, 23.29.1L-R174K and24.2.1, but the changes may represent conservative amino acidsubstitutions at such position(s) relative to the amino acid in thereference antibody. For example, if a particular position in one ofthese antibodies is changed relative to germline and is glutamate, onemay conservatively substitute aspartate at that position. Similarly, ifan amino acid substitution compared to germline is serine, one mayconservatively substitute threonine for serine at that position.Conservative amino acid substitutions are discussed supra.

In some embodiments, the light chain of the human anti-CD40 antibodycomprises the amino acid sequence that is the same as the amino acidsequence of the V_(L) of antibody 3.1.1 (SEQ. ID NO: 4), 3.1.1L-L4M-L83V (SEQ ID NO: 94), 7.1.2 (SEQ. ID NO: 12), 10.8.3 (SEQ. ID NO:20), 15.1.1 (SEQ. ID NO: 28), 21.4.1(SEQ. ID NO:), 21.2.1 (SEQ. ID NO:36), 21.4.1 (SEQ ID NO: 44), 22.1.1 (SEQ. ID NO: 52), 23.5.1 (SEQ. IDNO: 60), 23.28.1 (SEQ. ID NO: 68), 23.28.1L-C92A (SEQ. ID NO: 100),23.29.1 (SEQ. ID NO: 76), 23.29.1L-R174K (SEQ ID NO: 102) or 24.2.1(SEQ. ID NO: 84), or said amino acid sequence having up to 1, 2, 3, 4,6, 8 or 10 conservative amino acid substitutions and/or a total of up to3 non-conservative amino acid substitutions.

In some embodiments, the light chain of the anti-CD40 antibody comprisesat least the light chain CDR2, and may also comprise the CDR1 and CDR3regions of a germline sequence, as described herein. In anotherembodiment, the light chain may comprise a CDR1 and CDR2 of an antibodyindependently selected from 3.1.1, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3,15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1, 23.28.1L-C92A,23.29.1 and 24.2.1, or CDR regions each having less than 8, less than 6,less than 4 or less than 3 conservative amino acid substitutions and/ora total of three or fewer non-conservative amino acid substitutions. Inother embodiments, the light chain of the anti-CD40 antibody comprisesat least the light chain CDR2, and may also comprise the CDR1 and CDR3regions, each of which are independently selected from the CDR1 and CDR3regions of an antibody having a light chain variable region comprisingthe amino acid sequence selected from SEQ ID NOS: 4, 12, 20, 28, 36, 44,52, 60, 68, 76, 84, 94 or 100, or encoded by a nucleic acid moleculeselected from SEQ ID NOS: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 93or 99.

With regard to the heavy chain, in some embodiments, the variable regionof the heavy chain amino acid sequence is encoded in part by a humanV_(H) 3-30⁺, V_(H) 4-59, V_(H)1-02, V_(H) 4.35 or V_(H) 3-30.3 gene. Insome embodiments, the V_(H) of the anti-CD40 antibody contains one ormore amino acid substitutions, deletions or insertions (additions)relative to the germline amino acid sequence. In some embodiments, thevariable domain of the heavy chain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17 or 18 mutations from the germline aminoacid sequence. In some embodiments, the mutation(s) are non-conservativesubstitutions compared to the germline amino acid sequence. In someembodiments, the mutations are in the CDR regions of the heavy chain. Insome embodiments, the amino acid changes are made at one or more of thesame positions as the mutations from germline in any one or more of theV_(H) of antibodies 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 7.1.2,10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23.25.1,23.28.1, 23.28.1H-D16E, 23.29.1 and 24.2.1. In other embodiments, theamino acid changes are at one or more of the same positions but involvea different mutation than in the reference antibody.

In some embodiments, the heavy chain comprises an amino acid sequence ofthe variable domain (V_(H)) of antibody 3.1.1 (SEQ ID NO: 2),3.1.1H-A78T (SEQ ID NO: 90), 3.1.1H-A78T-V88A-V97A (SEQ ID NO: 92),7.1.2 (SEQ ID NO: 10), 10.8.3 (SEQ ID NO: 18), 15.1.1 (SEQ ID NO: 26),21.2.1 (SEQ ID NO: 34), 21.4.1 (SEQ ID NO: 42), 22.1.1 (SEQ ID NO: 50),22.1.1H-C109A (SEQ ID NO: 96), 23.5.1 (SEQ ID NO: 58), 23.28.1 (SEQ IDNO: 66), 23.28.1H-D16E (SEQ ID NO: 98), 23.29.1 (SEQ ID NO: 74) and24.2.1(SEQ ID NO: 82), or said amino acid sequence having up to 1, 2, 3,4, 6, 8 or 10 conservative amino acid substitutions and/or a total of upto 3 non-conservative amino acid substitutions.

In some embodiments, the heavy chain comprises the heavy chain CDR1,CDR2 and CDR3 regions of antibody 3.1.1, 3.1.1H-A78T,3.1.1H-A78T-V88A-V97A, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1,22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1H-D16E, 23.29.1 and24.2.1 (as shown in FIGS. 1D-1H or 2D-2H), or said CDR regions eachhaving less than 8, less than 6, less than 4, or less than 3conservative amino acid substitutions and/or a total of three or fewernon-conservative amino acid substitutions.

In some embodiments, the heavy chain comprises a CDR3, and may alsocomprise the CDR1 and CDR2 regions of a germline sequence, as describedabove, or may comprise a CDR1 and CDR2 of an antibody, each of which areindependently selected from an antibody comprising a heavy chain of anantibody selected from 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 7.1.2,10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23.25.1,23.28.1, 23.28.1H-D16E, 23.29.1 and 24.2.1. In another embodiment, theheavy chain comprises a CDR3, and may also comprise the CDR1 and CDR2regions, each of which are independently selected from a CDR1 and CDR2region of a heavy chain variable region comprising an amino acidsequence selected from SEQ ID NOS: 2, 10, 18, 26, 34, 42, 50, 58, 66,74, 82, 90, 92, 96 or 98 (as shown in FIGS. 1D-1H or FIGS. 2D-2H)orencoded by a nucleic acid sequence selected from SEQ ID NOS: 1, 9, 17,25, 33, 41, 49, 57, 65, 73, 81, 89, 91, 95 or 97. In another embodiment,the antibody comprises a heavy chain as disclosed above and a lightchain as disclosed above.

One type of amino acid substitution that may be made is to change one ormore cysteines in the antibody, which may be chemically reactive, toanother residue, such as, without limitation, alanine or serine. In oneembodiment, the cysteine substitution is made in a framework region of avariable domain or in the constant domain of an antibody. In anotherembodiment, the cysteine is in a non-canonical region of the antibody.Another type of amino acid substitution that may be made is to changeany potential proteolytic sites in the antibody, particularly those thatare in a framework region of a variable domain, in the constant domainof an antibody, or in a non-canonical region of the antibodySubstitution of cysteine residues and removal of proteolytic sites maydecrease the risk of any heterogeneity in the antibody product and thusincrease its homogeneity. Another type of amino acid substitution is toeliminate asparagine-glycine pairs, which form potential deamidationsites, by altering one or both of the residues. This is preferably donein framework regions, the constant domain or non-canonical regions ofthe antibody.

Activation of CD40 by Anti-CD40 Antibody

Another aspect of the present invention involves an anti-CD40 antibodythat is an activating antibody, i.e., a CD40 agonist. An activatingantibody amplifies or substitutes for the effects of CD40L on CD40. Insome embodiments, the activating antibody is essentially a mimic ofCD40L, and competes with CD40L for binding to CD40. In some embodiments,the antibody does not compete with CD40L for binding to CD40, butamplifies the effect of CD40L binding to CD40. In some embodiments, theanti-CD40 antibody activates CD40 in the presence or absence of CD40L.

Inhibition of Tumor Growth In Vivo by Anti-CD40 Antibodies

According to some embodiments, the invention provides an anti-CD40antibody that inhibits the proliferation of tumor cells in vitro ortumor growth in vivo.

In some embodiments, the antibody inhibits tumor growth by at least 50%,55%, 60%, 65%, 70%, 75%. In some embodiments, the antibody inhibitstumor growth by 75%. In one embodiment, the inhibition of tumor growthis detectable 14 days after initial treatment with the antibody. Inother embodiments, the inhibition of tumor growth is detectable 7 daysafter initial treatment with the antibody. In some embodiments, anotherantineoplastic agent is administered to the animal with the anti-CD40antibody. In some embodiments, the antineoplastic agent further inhibitstumor growth. In some embodiments, the antineoplastic agent isadriamycin or taxol. In some embodiments, the co-administration of anantineoplastic agent and the anti-CD40 antibody inhibits tumor growth byat least 50%, after a period of 22-24 days from initiation of treatmentcompared to tumor growth on an untreated animal.

Induction of Apoptosis by Anti-CD40 Antibodies

Another aspect of the invention provides an anti-CD40 antibody thatinduces cell death of CD40 positive cells. In some embodiments, theantibody causes apoptosis of CD40 positive cells either in vivo or invitro.

Enhancement of Expression of Cell Surface Molecules

In some embodiments, the anti-CD40 antibody enhances the expression of Bcell surface molecules, including but not limited to ICAM, MHC-II, B7-2,CD71, CD23 and CD83. In some embodiments, 1 μg/ml of the antibodyenhances ICAM expression in a whole blood B-cell surface moleculeup-regulation assay by at least 2 fold, or more preferably by at least 4fold. In some embodiments, 1 μg/ml of the antibody enhances MHC-IIexpression in a whole blood B-cell surface molecule upregulation assayby at least 2 fold, or more preferably by at least 3 fold. In someembodiments, 1 μg/ml of the antibody enhances CD23 expression in wholeblood B-cell surface molecule up-regulation assay by at least 2 fold, ormore preferably by at least 5 fold. See, e.g., Example VII, Table 25.

In some embodiments, the anti-CD40 antibody enhances the expression ofdendritic cell surface molecules including but not limited to MHC-II,ICAM, B7-2, CD83 and B7-1. In some embodiments the range of upregulationis similar to the range of upregulation observed in B cells. See, e.g.,Tables 25 and 26, infra. In some embodiments, the antibodypreferentially upregulates the expression of dendritic cell surfacemolecules, such as B7-2 and MHC-II, compared to B cell expression ofthese molecules. See, e.g., Table 27.

Enhancement of Secretion of Cellular Cytokines

In some embodiments the antibody enhances cellular secretion ofcytokines including but not limited to IL-8, IL-12, IL-15, IL-18 andIL-23.

In some embodiments the antibody enhances cytokine secretion bydendritic cells and adherent monocytes. In some embodiments cytokineproduction is further enhanced by co-stimulation with one or more ofLPS, IFN-γ or IL-1β. In yet another aspect of the invention, theantibody with LPS co-stimulation enhances IL-12p70 production in adendritic cell assay with an EC₅₀ of about 0.48 μg/ml. In someembodiments, the antibody enhances IL-12p40 production in dendriticcells with an EC₅₀ of about 0.21 μg/ml. (See, e.g., Example VIII.)

In some embodiments, the antibody enhances secretion of IFN-gamma by Tcells in an allogenic T cell/dendritic cell assay, as described inExample VIII. In some embodiments, the antibody enhances IFN-gammasecretion in an allogenic T cell/dendritic cell assay with an EC₅₀ ofabout 0.3 μg/ml. In some embodiments, the antibody enhances IFN-gammasecretion in an allogenic T cell/dendritic cell assay with an EC₅₀ ofabout 0.2 μg/ml. In one embodiment, the antibody enhances IFN-gammasecretion in an allogenic T cell/dendritic cell assay with an EC₅₀ Ofabout 0.03 μg/ml.

Methods of Producing Antibodies and Antibody-Producing Cell Lines

Immunization

In some embodiments, human antibodies are produced by immunizing anon-human animal comprising in its genome some or all of humanimmunoglobulin heavy chain and light chain loci with a CD40 antigen. Ina preferred embodiment, the non-human animal is a XenoMouse™ animal.

XenoMouse™ mice are engineered mouse strains that comprise largefragments of human immunoglobulin heavy chain and light chain loci andare deficient in mouse antibody production. See, e.g., Green et al.,Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771, 5,939,598,5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364,6,162,963 and 6,150,584. See also WO 91/10741, WO 94/02602, WO 96/34096,WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO99/53049, WO 00/09560, and WO 00/037504.

In another aspect, the invention provides a method for making anti-CD40antibodies from non-human, non-mouse animals by immunizing non-humantransgenic animals that comprise human immunoglobulin loci with a CD40antigen. One can produce such animals using the methods described in theabove-cited documents. The methods disclosed in these documents can bemodified as described in U.S. Pat. No. 5,994,619. In preferredembodiments, the non-human animals are rats, sheep, pigs, goats, cattleor horses.

XenoMouse™ mice produce an adult-like human repertoire of fully humanantibodies and generate antigen-specific human antibodies. In someembodiments, the XenoMouse™ mice contain approximately 80% of the humanantibody V gene repertoire through introduction of megabase sized,germline configuration yeast artificial chromosome (YAC) fragments ofthe human heavy chain loci and kappa light chain loci. See Mendez etal., Nature Genetics 15:146-156 (1997), Green and Jakobovits, J. Exp.Med. 188:483-495 (1998), and WO 98/24893, the disclosures of which arehereby incorporated by reference.

In some embodiments, the non-human animal comprising humanimmunoglobulin genes are animals that have a human immunoglobulin“minilocus”. In the minilocus approach, an exogenous Ig locus ismimicked through the inclusion of individual genes from the Ig locus.Thus, one or more V_(H) genes, one or more D_(H) genes, one or moreJ_(H) genes, a mu constant domain, and a second constant domain(preferably a gamma constant domain) are formed into a construct forinsertion into an animal. This approach is described, inter alia, inU.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425,5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205,5,721,367, 5,789,215, and 5,643,763, hereby incorporated by reference.

An advantage of the minilocus approach is the rapidity with whichconstructs including portions of the Ig locus can be generated andintroduced into animals. However, a potential disadvantage of theminilocus approach is that there may not be sufficient immunoglobulindiversity to support full B-cell development, such that there may belower antibody production.

In another aspect, the invention provides a method for making humanizedanti-CD40 antibodies. In some embodiments, non-human animals areimmunized with a CD40 antigen as described below under conditions thatpermit antibody production. Antibody-producing cells are isolated fromthe animals, fused with myelomas to produce hybridomas, and nucleicacids encoding the heavy and light chains of an anti-CD40 antibody ofinterest are isolated. These nucleic acids are subsequently engineeredusing techniques known to those of skill in the art and as describedfurther below to reduce the amount of non-human sequence, i.e., tohumanize the antibody to reduce the immune response in humans

In some embodiments, the CD40 antigen is isolated and/or purified CD40.In a preferred embodiment, the CD40 antigen is human CD40. In someembodiments, the CD40 antigen is a fragment of CD40. In someembodiments, the CD40 fragment is the extracellular domain of CD40. Insome embodiments, the CD40 fragment comprises at least one epitope ofCD40. In other embodiments, the CD40 antigen is a cell that expresses oroverexpresses CD40 or an immunogenic fragment thereof on its surface. Insome embodiments, the CD40 antigen is a CD40 fusion protein.

Immunization of animals can be by any method known in the art. See,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: ColdSpring Harbor Press, 1990. Methods for immunizing non-human animals suchas mice, rats, sheep, goats, pigs, cattle and horses are well known inthe art. See, e.g., Harlow and Lane, supra, and U.S. Pat. No. 5,994,619.In a preferred embodiment, the CD40 antigen is administered with anadjuvant to stimulate the immune response. Exemplary adjuvants includecomplete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) orISCOM (immunostimulating complexes). Such adjuvants may protect thepolypeptide from rapid dispersal by sequestering it in a local deposit,or they may contain substances that stimulate the host to secretefactors that are chemotacetic for macrophages and other components ofthe immune system. Preferably, if a polypeptide is being administered,the immunization schedule will involve two or more administrations ofthe polypeptide, spread out over several weeks.

Example I describes the production of anti-CD40 monoclonal antibodies.

Production of Antibodies and Antibody-Producing Cell Lines

After immunization of an animal with a CD40 antigen, antibodies and/orantibody-producing cells can be obtained from the animal. In someembodiments, anti-CD40 antibody-containing serum is obtained from theanimal by bleeding or sacrificing the animal. The serum may be used asit is obtained from the animal, an immunoglobulin fraction may beobtained from the serum, or the anti-CD40 antibodies may be purifiedfrom the serum. It is well known to one of ordinary skill in the artthat serum or immunoglobulins obtained in this manner will bepolyclonal. The disadvantage is using polyclonal antibodies preparedfrom serum is that the amount of antibodies that can be obtained islimited and the polyclonal antibody has a heterogeneous array ofproperties.

In some embodiments, antibody-producing immortalized cell lines areprepared from cells isolated from the immunized animal. Afterimmunization, the animal is sacrificed and lymph node and/or splenic Bcells are immortalized. Methods of immortalizing cells include, but arenot limited to, transferring them with oncogenes, inflecting them withthe oncogenic virus cultivating them under conditions that select forimmortalized cells, subjecting them to carcinogenic or mutatingcompounds, fusing them with an immortalized cell, e.g., a myeloma cell,and inactivating a tumor suppressor gene. See, e.g., Harlow and Lane,supra. If fusion with myeloma cells is used, the myeloma cellspreferably do not secrete immunoglobulin polypeptides (a non-secretorycell line). Immortalized cells are screened using CD40, a portionthereof, or a cell expressing CD40. In a preferred embodiment, theinitial screening is performed using an enzyme-linked immunoassay(ELISA) or a radioimmunoassay. An example of ELISA screening is providedin WO 00/37504, herein incorporated by reference.

Anti-CD40 antibody-producing cells, e.g., hybridomas, are selected,cloned and further screened for desirable characteristics, includingrobust growth, high antibody production and desirable antibodycharacteristics, as discussed further below. Hybridomas can be expandedin vivo in syngeneic animals, in animals that lack an immune system,e.g., nude mice, or in cell culture in vitro. Methods of selecting,cloning and expanding hybridomas are well known to those of ordinaryskill in the art.

In a preferred embodiment, the immunized animal is a non-human animalthat expresses human immunoglobulin genes and the splenic B cells arefused to a myeloma cell line from the same species as the non-humananimal. In a more preferred embodiment, the immunized animal is aXENOMOUSE animal and the myeloma cell line is a non-secretory mousemyeloma. In an even more preferred embodiment, the myeloma cell line isP3-X63-AG8.653. See, e.g., Example I.

In another aspect, the invention provides hybridomas that produce anhuman anti-CD40 antibody. In a preferred embodiment, the hybridomas aremouse hybridomas, as described above. In other embodiments, thehybridomas are produced in a non-human, non-mouse species such as rats,sheep, pigs, goats, cattle or horses. In another embodiment, thehybridomas are human hybridomas.

Nucleic Acids, Vectors, Host Cells and Recombinant Methods of MakingAntibodies

Nucleic Acids

The present invention also encompasses nucleic acid molecules encodinganti-CD40 antibodies. In some embodiments, different nucleic acidmolecules encode a heavy chain and a light chain of an anti-CD40immunoglobulin. In other embodiments, the same nucleic acid moleculeencodes a heavy chain and a light chain of an anti-CD40 immunoglobulin.

In some embodiments, the nucleic acid molecule encoding the variabledomain of the light chain comprises a human A3/A19 (DPK-15), L5 (DP5) orA27 (DPK-22) Vκ gene sequence or a sequence derived therefrom. In someembodiments, the nucleic acid molecule comprises a nucleotide sequenceof a A3/A19 Vκ gene and a Jκ1, Jκ2 or Jκ3 gene or sequences derivedtherefrom. In some embodiments, the nucleic acid molecule comprises anucleotide sequence of an L5 Vκ gene and a Jκ4 gene. In someembodiments, the nucleic acid molecule comprises a nucleotide sequenceof a A27 Vκ gene and a Jκ3 gene.

In some embodiments, the nucleic acid molecule encoding the light chain,encodes an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 mutations from the germline amino acid sequence. In some embodiments,the nucleic acid molecule comprises a nucleotide sequence that encodes aV_(L) amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10non-conservative amino acid substitutions and/or 1, 2 or 3non-conservative substitutions compared to the germline sequence.Substitutions may be in the CDR regions, the framework regions or in theconstant domain.

In some embodiments, the nucleic acid molecule encoding the variabledomain of the light chain (V_(L)) encodes a V_(L) amino acid sequencecomprising one or more mutations compared to the germline sequence thatare identical to the mutations found in the V_(L) of one of theantibodies 3.1.1, 3.1. IL-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1,21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1, 23.28.1L-C92A, 23.29.1 and24.2.1. In some embodiments, the nucleic acid molecule encodes at leastthree amino acid mutations compared to the germline sequence found inthe V_(L) of one of the antibodies 3.1.1, 3.1.1 L-L4M-L83V, 7.1.2,10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1,23.28.1L-C92A, 23.29.1 and 24.2.1.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the V_(L) amino acid sequence of monoclonalantibody 3.1.1 (SEQ ID NO: 4), 3.1.1L-L4M-L83V (SEQ ID NO: 94), 7.1.2(SEQ ID NO: 12), 10.8.3 (SEQ ID NO: 20), 15.1.1 (SEQ ID NO: 28), 21.2.1(SEQ ID NO: 36), 2.1.4.1 (SEQ ID NO: 44), 22.1.1 (SEQ ID NO: 52), 23.5.1(SEQ ID NO: 60), 23.28.1 (SEQ ID NO: 68), 23.28.1L-C92A (SEQ ID NO:100), 23.29.1 (SEQ ID NO: 76) or 24.2.1 (SEQ ID NO: 84), or a portionthereof. In some embodiments, said portion comprises at least the CDR3region. In some embodiments, the nucleic acid encodes the amino acidsequence of the light chain CDRs of said antibody. In some embodiments,said portion is a contiguous portion comprising CDR1-CDR3.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the amino acid sequence of one of SEQ ID NOS: 4,12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 94 or 100, or said sequencelacking the signal sequence. In some preferred embodiments, the nucleicacid molecule comprises the nucleotide sequence of SEQ ID NOS: 3, 11,19, 27, 35, 43, 51, 59, 67, 75, 83, 93 or 99, or a portion thereof, saidsequences optionally lacking the signal sequence.

In some embodiments, said portion encodes a V_(L) region. In someembodiments, said portion encodes at least the CDR2 region. In someembodiments, the nucleic acid encodes the amino acid sequence of thelight chain CDRs of said antibody. In some embodiments, said portionencodes a contiguous region from CDR1-CDR3.

In some embodiments, the nucleic acid molecule encodes a V_(L) aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to a V_(L) amino acid sequence of any one of antibodies3.1.1, 3.1. IL-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1,23.5.1, 23.25.1, 23.28.1, 23.28.1L-C92A, 23.29.1 or 24.2.1, or a V_(L)amino acid sequence of any one of SEQ ID NOS: 4, 12, 20, 28, 36, 44, 52,60, 68, 76, 84, 94 or 100. Nucleic acid molecules of the inventioninclude nucleic acids that hybridize under highly stringent conditions,such as those described above, to a nucleic acid sequence encoding theamino acid sequence of SEQ ID NOS: 4, 12, 20, 28, 36, 44, 52, 60, 68,76, 84, 94 or 100, or that has the nucleic acid sequence of SEQ ID NOS:3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 93 or 99.

In another embodiment, the nucleic acid encodes a full-length lightchain of an antibody selected from 3.1.1, 3.1.1L-L4M-L83V, 7.1.2,10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1,23.28.1L-C92A, 23.29.1, 23.29.1L-R174K or 24.2.1, or a light chaincomprising the amino acid sequence of SEQ ID NOS: 8, 16, 24, 32, 40, 48,56, 64, 72, 80, 88, 94, 100 or 102, or a light chain comprising amutation, such as one disclosed herein. Further, the nucleic acid maycomprise the nucleotide sequence of SEQ ID NOS: 7, 15, 23, 31, 39, 47,55, 63, 71, 79 or 87, or a nucleic acid molecule encoding a light chaincomprise a mutation, such as one disclosed herein.

In another preferred embodiment, the nucleic acid molecule encodes thevariable domain of the heavy chain (V_(H)) that comprises a human 3-30+,4-59, 1-02, 4.35 or 3-30.3 V_(H) gene sequence or a sequence derivedtherefrom. In various embodiments, the nucleic acid molecule comprises ahuman 3-30+V_(H) gene, a D4 (DIR3) gene and a human J_(H)6 gene; a human3-30+V_(H) gene, a human D1-26 (DIR5) gene and a human J_(H)6 gene; ahuman 4.35 V_(H) gene, a human DIR3 gene and a human J_(H)6 gene; ahuman 4-59 V_(H) gene, a human D4-23 gene and a human J_(H)4 gene; ahuman 1-02 V_(H) gene, a human DLR1 gene and a human J_(H) ⁴ gene; ahuman 3-30+V_(H) gene, a human D6-19 (DIR3) gene and a human J_(H) ⁴gene; a human 3-30+V_(H) gene, a human D1-1 gene and a human J_(H)6gene; a human 3-30+V_(H) gene, a human D4-17 gene and a human J_(H)6gene; a human 3-30.3 V_(H) gene, a human D4-17 gene and a human J_(H)6gene; a human 4-59 V_(H) gene, a human D4-17 (DIR1) gene and a humanJ_(H)5 gene, or sequence derived from the human genes.

In some embodiments, the nucleic acid molecule encodes an amino acidsequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17 or 18 mutations compared to the germline amino acid sequence ofthe human V, D or J genes. In some embodiments, said mutations are inthe V_(H) region. In some embodiments, said mutations are in the CDRregions.

In some embodiments, the nucleic acid molecule encodes one or more aminoacid mutations compared to the germline sequence that are identical toamino acid mutations found in the V_(H) of monoclonal antibody 3.1.1,3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 7.1.2, 10.8.3, 15.1.1, 21.4.1,21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1H-D16E,23.29.1 or 24.2.1. In some embodiments, the nucleic acid encodes atleast three amino acid mutations compared to the germline sequences thatare identical to at least three amino acid mutations found in one of theabove-listed monoclonal antibodies.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes at least a portion of the V_(H) amino acidsequence of antibody 3.1.1 (SEQ ID NO: 2), 3.1.1H-A78T (SEQ ID NO: 90),3.1.1H-A78T-V88A-V97A (SEQ ID NO: 92), 7.1.2 (SEQ ID NO: 10), 10.8.3(SEQ ID NO: 18), 15.1.1 (SEQ ID NO: 26), 21.2.1 (SEQ ID NO: 34), 21.4.1(SEQ ID NO: 42), 22.1.1 (SEQ ID NO: 50), 22.1.1H-C109A (SEQ ID NO: 96),23.5.1 (SEQ ID NO: 58), 23.28.1 (SEQ ID NO: 66), 23.28.1H-D16E (SEQ IDNO: 98), 23.29.1 (SEQ ID NO: 74) or 24.2.1 (SEQ ID NO: 82), or saidsequence having conservative amino acid mutations and/or a total ofthree or fewer non-conservative amino acid substitutions. In variousembodiments the sequence encodes one or more CDR regions, preferably aCDR3 region, all three CDR regions, a contiguous portion includingCDR1-CDR3, or the entire V_(H) region, with or without a signalsequence.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the amino acid sequence of one of SEQ ID NOS: 2,10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 92, 96 or 98, or saidsequence lacking the signal sequence. In some preferred embodiments, thenucleic acid molecule comprises at least a portion of the nucleotidesequence of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 91,95 or 97, or said sequence lacking the signal sequence. In someembodiments, said portion encodes the V_(H) region (with or without asignal sequence), a CDR3 region, all three CDR regions, or a contiguousregion including CDR1-CDR3.

In some embodiments, the nucleic acid molecule encodes a V_(H) aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to the V_(H) amino acid sequences shown in FIGS. 1A-1C or2A-2C or to a V_(H) amino acid sequence of any one of SEQ ID NOS: 2, 10,18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 92, 96 or 98. Nucleic acidmolecules of the invention include nucleic acids that hybridize underhighly stringent conditions, such as those described above, to a nucleicacid sequence encoding the amino acid sequence of SEQ ID NOS: 2, 10, 18,26, 34, 42, 50, 58, 66, 74, 82, 90, 92, 96 or 98, or that has thenucleic acid sequence of SEQ ID NOS: 1, 9, 17, 25, 33, 41, 49, 57, 65,73, 81, 89, 91, 95 or 97. Nucleic acid molecule of the invention includenucleic acid molecule that hybridize under highly stringent conditions,such as those described above, to a nucleic acid sequence encoding aV_(H) described immediately above.

In another embodiment, the nucleic acid encodes a full-length heavychain of an antibody selected from 3.1.1, 3.1.1H-A78T,3.1.1H-A78T-V88A-V97A, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1,22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1H-D16E, 23.29.1 and24.2.1, or a heavy chain having the amino acid sequence of SEQ ID NOS:6, 14, 22, 30, 38, 46, 54, 62, 70, 78 or 86, or a heavy chain comprisinga mutation, such as one of the mutations discussed herein Further, thenucleic acid may comprise the nucleotide sequence of SEQ ID NOS: 5, 13,21, 29, 37, 45, 53, 61, 69, 77, 85 or 89, or a nucleic acid moleculeencoding a heavy chain comprising a mutation, such as one of themutations discussed herein.

A nucleic acid molecule encoding the heavy or entire light chain of ananti-CD40 antibody or portions thereof can be isolated from any sourcethat produces such antibody. In various embodiments, the nucleic acidmolecules are isolated from a B cell isolated from an animal immunizedwith CD40 or from an immortalized cell derived from such a B cell thatexpresses an anti-CD40 antibody. Methods of isolating mRNA encoding anantibody are well-known in the art. See, e.g., Sambrook et al. The mRNAmay be used to produce cDNA for use in the polymerase chain reaction(PCR) or cDNA cloning of antibody genes. In a preferred embodiment, thenucleic acid molecule is isolated from a hybridoma that has as one ofits fusion partners a human immunoglobulin-producing cell from anon-human transgenic animal. In an even more preferred embodiment, thehuman immunoglobulin producing cell is isolated from a XenoMouse™animal. In another embodiment, the human immunoglobulin-producing cellis from a non-human, non-mouse transgenic animal, as described above. Inanother embodiment, the nucleic acid is isolated from a non-human,non-transgenic animal. The nucleic acid molecules isolated from anon-human, non-transgenic animal may be used, e.g., for humanizedantibodies.

In some embodiments, a nucleic acid encoding a heavy chain of ananti-CD40 antibody of the invention can comprise a nucleotide sequenceencoding a V_(H) domain of the invention joined in-frame to a nucleotidesequence encoding a heavy chain constant domain from any source.Similarly, a nucleic acid molecule encoding a light chain of ananti-CD40 antibody of the invention can comprise a nucleotide sequenceencoding a V_(L) domain of the invention joined in-frame to a nucleotidesequence encoding a light chain constant domain from any source.

In a further aspect of the invention, nucleic acid molecules encodingthe variable domain of the heavy (V_(H)) and light (V_(L)) chains are“converted” to full-length antibody genes. In one embodiment, nucleicacid molecules encoding the V_(H) or V_(L) domains are converted tofull-length antibody genes by insertion into an expression vectoralready encoding heavy chain constant or light chain constant domains,respectively, such that the V_(H) segment is operatively linked to theC_(H) segment(s) within the vector, and the V_(L) segment is operativelylinked to the C_(L) segment within the vector. In another embodiment,nucleic acid molecules encoding the V_(H) and/or V_(L) domains areconverted into full-length antibody genes by linking, e.g., ligating, anucleic acid molecule encoding a V_(H) and/or V_(L) domains to a nucleicacid molecule encoding a C_(H) and/or C_(L) domain using standardmolecular biological techniques. Nucleic acid sequences of human heavyand light chain immunoglobulin constant domain genes are known in theart. See, e.g., Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed., NIH Publ. No. 91-3242, 1991. Nucleic acid moleculesencoding the full-length heavy and/or light chains may then be expressedfrom a cell into which they have been introduced and the anti-CD40antibody isolated.

The nucleic acid molecules may be used to recombinantly express largequantities of anti-CD40 antibodies. The nucleic acid molecules also maybe used to produce chimeric antibodies, bispecific antibodies, singlechain antibodies, immunoadhesins, diabodies, mutated antibodies andantibody derivatives, as described further below. If the nucleic acidmolecules are derived from a non-human, non-transgenic animal, thenucleic acid molecules may be used for antibody humanization, also asdescribed below.

In another embodiment, a nucleic acid molecule of the invention is usedas a probe or PCR primer for a specific antibody sequence. For instance,the nucleic acid can be used as a probe in diagnostic methods or as aPCR primer to amplify regions of DNA that could be used, inter alia, toisolate additional nucleic acid molecules encoding variable domains ofanti-CD40 antibodies. In some embodiments, the nucleic acid moleculesare oligonucleotides. In some embodiments, the oligonucleotides are fromhighly variable regions of the heavy and light chains of the antibody ofinterest. In some embodiments, the oligonucleotides encode all or a partof one or more of the CDRs of antibody 3.1.1, 3.1.1H-A78T,3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1,21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1H-D116E,23.28.1 L-C92A, 23.29.1 or 24.2.1.

Vectors

The invention provides vectors comprising nucleic acid molecules thatencode the heavy chain of an anti-CD40 antibody of the invention or anantigen-binding portion thereof. The invention also provides vectorscomprising nucleic acid molecules that encode the light chain of suchantibodies or antigen-binding portion thereof. The invention furtherprovides vectors comprising nucleic acid molecules encoding fusionproteins, modified antibodies, antibody fragments, and probes thereof.

In some embodiments, the anti-CD40 antibodies, or antigen-bindingportions of the invention are expressed by inserting DNAs encodingpartial or full-length light and heavy chains, obtained as describedabove, into expression vectors such that the genes are operativelylinked to necessary expression control sequences such as transcriptionaland translational control sequences. Expression vectors includeplasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV),plant viruses such as cauliflower mosaic virus, tobacco mosaic virus,cosmids, YACs, EBV derived episomes, and the like. The antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevectors. In a preferred embodiment, both genes are inserted into thesame expression vector. The antibody genes are inserted into theexpression vector by standard methods (e.g., ligation of complementaryrestriction sites on the antibody gene fragment and vector, or blunt endligation if no restriction sites are present).

A convenient vector is one that encodes a functionally complete humanC_(H) or C_(L) immunoglobulin sequence, with appropriate restrictionsites engineered so that any V_(H) or V_(L) sequence can easily beinserted and expressed, as described above. In such vectors, splicingusually occurs between the splice donor site in the inserted J regionand the splice acceptor site preceding the human C domain, and also atthe splice regions that occur within the human C_(H) exons.Polyadenylation and transcription termination occur at nativechromosomal sites downstream of the coding regions. The recombinantexpression vector also can encode a signal peptide that facilitatessecretion of the antibody chain from a host cell. The antibody chaingene may be cloned into the vector such that the signal peptide islinked in-frame to the amino terminus of the immunoglobulin chain. Thesignal peptide can be an immunoglobulin signal peptide or a heterologoussignal peptide (i.e., a signal peptide from a non-immunoglobulinprotein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. It will beappreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from retroviral LTRs, cytomegalovirus(CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (suchas the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus majorlate promoter (AdMLP)), polyoma and strong mammalian promoters such asnative immunoglobulin and actin promoters. For further description ofviral regulatory elements, and sequences thereof, see e.g., U.S. Pat.No. 5,168,062, U.S. Pat. No. 4,510,245 and U.S. Pat. No. 4,968,615.Methods for expressing antibodies in plants, including a description ofpromoters and vectors, as well as transformation of plants is known inthe art. See, e.g., U.S. Pat. No. 6,517,529, herein incorporated byreference. Methods of expressing polypeptides in bacterial cells orfungal cells, e.g., yeast cells, are also well known in the art.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Preferred selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification), the neo gene (for G418selection), and the glutamate synthetase gene.

Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein

Nucleic acid molecules encoding anti-CD40 antibodies and vectorscomprising these nucleic acid molecules can be used for transfection ofa suitable mammalian, plant, bacterial or yeast host cell.Transformation can be by any known method for introducingpolynucleotides into a host cell. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei. In addition, nucleicacid molecules may be introduced into mammalian cells by viral vectors.Methods of transforming cells are well known in the art. See, e.g., U.S.Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patentsare hereby incorporated herein by reference). Methods of transformingplant cells are well known in the art, including, e.g.,Agrobacterium-mediated transformation, biolistic transformation, directinjection, electroporation and viral transformation. Methods oftransforming bacterial and yeast cells are also well known in the art.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC). These include, inter alia,Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a numberof other cell lines. Cell lines of particular preference are selectedthrough determining which cell lines have high expression levels. Othercell lines that may be used are insect cell lines, such as Sf9 cells.When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods. Plant host cells include,e.g., Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc.Bacterial host cells include E. coli and Streptomyces species. Yeasthost cells include Schizosaccharomyces pombe, Saccharomyces cerevisiaeand Pichia pastoris.

Further, expression of antibodies of the invention (or other moietiestherefrom) from production cell lines can be enhanced using a number ofknown techniques. For example, the glutamine synthetase gene expressionsystem (the GS system) is a common approach for enhancing expressionunder certain conditions. The GS system is discussed in whole or part inconnection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997and European Patent Application No. 89303964.4.

It is likely that antibodies expressed by different cell lines or intransgenic animals will have different glycosylation from each other.However, all antibodies encoded by the nucleic acid molecules providedherein, or comprising the amino acid sequences provided herein are partof the instant invention, regardless of the glycosylation of theantibodies.

Transgenic Animals and Plants

Anti-CD40 antibodies of the invention also can be producedtransgenically through the generation of a mammal or plant that istransgenic for the immunoglobulin heavy and light chain sequences ofinterest and production of the antibody in a recoverable form therefrom.In connection with the transgenic production in mammals, anti-CD40antibodies can be produced in, and recovered from, the milk of goats,cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687,5,750,172, and 5,741,957. In some embodiments, non-human transgenicanimals that comprise human immunoglobulin loci are immunized with CD40or an immunogenic portion thereof, as described above. Methods formaking antibodies in plants are described, e.g., in U.S. Pat. No.6,046,037 and U.S. Pat. No. 5,959,177.

In some embodiments, non-human transgenic animals or plants are producedby introducing one or more nucleic acid molecules encoding an anti-CD40antibody of the invention into the animal or plant by standardtransgenic techniques. See Hogan and U.S. Pat. No. 6,417,429, supra. Thetransgenic cells used for making the transgenic animal can be embryonicstem cells or somatic cells. The transgenic non-human organisms can bechimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See,e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual2ed., Cold Spring Harbor Press (1999); Jackson et al., Mouse Geneticsand Transgenics: A Practical Approach, Oxford University Press (2000);and Pinkert, Transgenic Animal Technology: A Laboratory Handbook,Academic Press (1999). In some embodiments, the transgenic non-humananimals have a targeted disruption and replacement by a targetingconstruct that encodes a heavy chain and/or a light chain of interest.In a preferred embodiment, the transgenic animals comprise and expressnucleic acid molecules encoding heavy and light chains that specificallybind to CD40, preferably human CD40. In some embodiments, the transgenicanimals comprise nucleic acid molecules encoding a modified antibodysuch as a single-chain antibody, a chimeric antibody or a humanizedantibody. The anti-CD40 antibodies may be made in any transgenic animal.In a preferred embodiment, the non-human animals are mice, rats, sheep,pigs, goats, cattle or horses. The non-human transgenic animal expressessaid encoded polypeptides in blood, milk, urine, saliva, tears, mucusand other bodily fluids.

Phage Display Libraries

The invention provides a method for producing an anti-CD40 antibody orantigen-binding portion thereof comprising the steps of synthesizing alibrary of human antibodies on phage, screening the library with CD40 ora portion thereof, isolating phage that bind CD40, and obtaining theantibody from the phage. By way of example, one method for preparing thelibrary of antibodies for use in phage display techniques comprises thesteps of immunizing a non-human animal comprising human immunoglobulinloci with CD40 or an antigenic portion thereof to create an immuneresponse, extracting antibody producing cells from the immunized animal;isolating RNA from the extracted cells, reverse transcribing the RNA toproduce cDNA, amplifying the cDNA using a primer, and inserting the cDNAinto a phage display vector such that antibodies are expressed on thephage. Recombinant anti-CD40 antibodies of the invention may be obtainedin this way.

Recombinant anti-CD40 human antibodies of the invention can be isolatedby screening a recombinant combinatorial antibody library. Preferablythe library is a scFv phage display library, generated using human V_(L)and V_(H) cDNAs prepared from mRNA isolated from B cells. Methodologiesfor preparing and screening such libraries are known in the art. Thereare commercially available kits for generating phage display libraries(e.g., the Pharmacia Recombinant Phage Antibody System, catalog no.27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no.240612). There also are other methods and reagents that can be used ingenerating and screening antibody display libraries (see, e.g., U.S.Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO92/20791, WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs etal., Bio/Technology 9:1370-1372 (1991); Hay et al., Hum. Antibod.Hybridomas 3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989);McCafferty et al., Nature 348:552-554 (1990); Griffiths et al., EMBO J.12:725-734 (1993); Hawkins et al., J. Mol. Biol. 226:889-896 (1992);Clackson et al., Nature 352:624-628 (1991); Gram et al., Proc. Natl.Acad. Sci. USA 89:3576-3580 (1992); Garrad et al., Bio/Technology9:1373-1377 (1991); Hoogenboom et al., Nuc. Acid Res. 19:4133-4137(1991); and Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982(1991).

In one embodiment, to isolate a human anti-CD40 antibodies with thedesired characteristics, a human anti-CD40 antibody as described hereinis first used to select human heavy and light chain sequences havingsimilar binding activity toward CD40, using the epitope imprintingmethods described in PCT Publication No. WO 93/06213. The antibodylibraries used in this method are preferably scFv libraries prepared andscreened as described in PCT Publication No. WO 92/01047, McCafferty etal., Nature 348:552-554 (1990); and Griffiths et al., EMBO J. 12:725-734(1993). The scFv antibody libraries preferably are screened using humanCD40 as the antigen.

Once initial human V_(L) and V_(H) domains are selected, “mix and match”experiments are performed, in which different pairs of the initiallyselected V_(L) and V_(H) segments are screened for CD40 binding toselect preferred V_(L)/V_(H) pair combinations. Additionally, to furtherimprove the quality of the antibody, the V_(L) and V_(H) segments of thepreferred V_(L)/V_(H) pair(s) can be randomly mutated, preferably withinthe CDR3 region of V_(H) and/or V_(L), in a process analogous to the invivo somatic mutation process responsible for affinity maturation ofantibodies during a natural immune response. This in vitro affinitymaturation can be accomplished by amplifying V_(H) and V_(L) domainsusing PCR primers complimentary to the V_(H) CDR3 or V_(L) CDR3,respectively, which primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode V_(H) and V_(L) segments into which random mutationshave been introduced into the V_(H) and/or V_(L) CDR3 regions. Theserandomly mutated V_(H) and V_(L) segments can be rescreened for bindingto CD40.

Following screening and isolation of an anti-CD40 antibody of theinvention from a recombinant immunoglobulin display library, nucleicacids encoding the selected antibody can be recovered from the displaypackage (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can further be manipulated to create other antibodyforms of the invention, as described below. To express a recombinanthuman antibody isolated by screening of a combinatorial library, the DNAencoding the antibody is cloned into a recombinant expression vector andintroduced into a mammalian host cells, as described above.

Class Switching

Another aspect of the invention provides a method for converting theclass or subclass of an anti-CD40 antibody to another class or subclass.In some embodiments, a nucleic acid molecule encoding a V_(L) or V_(H)that does not include any nucleic acid sequences encoding C_(L) or C_(H)is isolated using methods well-known in the art. The nucleic acidmolecule then is operatively linked to a nucleic acid sequence encodinga C_(L) or C_(H) from a desired immunoglobulin class or subclass. Thiscan be achieved using a vector or nucleic acid molecule that comprises aC_(L) or C_(H) chain, as described above. For example, an anti-CD40antibody that was originally IgM can be class switched to an IgG.Further, the class switching may be used to convert one IgG subclass toanother, e.g., from IgG1 to IgG2. Another method for producing anantibody of the invention comprising a desired isotype comprises thesteps of isolating a nucleic acid encoding a heavy chain of an anti-CD40antibody and a nucleic acid encoding a light chain of an anti-CD40antibody, isolating the sequence encoding the V_(H) region, ligating theV_(H) sequence to a sequence encoding a heavy chain constant domain ofthe desired isotype, expressing the light chain gene and the heavy chainconstruct in a cell, and collecting the anti-CD40 antibody with thedesired isotype.

Deimmunized Antibodies

Another way of producing antibodies with reduced immunogenicity is thedeimmunization of antibodies. In another aspect of the invention, theantibody may be deimmunized using the techniques described in, e.g., PCTPublication Nos. WO98/52976 and WO00/34317 (which incorporated herein byreference in their entirety).

Mutated Antibodies

In another embodiment, the nucleic acid molecules, vectors and hostcells may be used to make mutated anti-CD40 antibodies. The antibodiesmay be mutated in the variable domains of the heavy and/or light chains,e.g., to alter a binding property of the antibody. For example, amutation may be made in one or more of the CDR regions to increase ordecrease the K_(D) of the antibody for CD40, to increase or decreaseK_(off), or to alter the binding specificity of the antibody. Techniquesin site-directed mutagenesis are well-known in the art. See, e.g.,Sambrook et al. and Ausubel et al., supra. In a preferred embodiment,mutations are made at an amino acid residue that is known to be changedcompared to germline in a variable domain of an anti-CD40 antibody. Inanother embodiment, one or more mutations are made at an amino acidresidue that is known to be changed compared to the germline in a CDRregion or framework region of a variable domain, or in a constant domainof a monoclonal antibody 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A,3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1,22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1, 23.28.1H-D16E, 23.28.1L-C92A,23.29.1, 23.29.1L-R174K and 24.2.1. In another embodiment, one or moremutations are made at an amino acid residue that is known to be changedcompared to the germline in a CDR region or framework region of avariable domain of an amino acid sequence selected from SEQ ID NOS: 4,12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 94, 100, 102, 2, 10, 18, 26, 34,42, 50, 58, 66, 74, 82, 90, 92, 96, 98, 100 or 102, or whose nucleicacid sequence is presented in SEQ ID NOS: 3, 11, 19, 27, 35, 43, 51, 59,67, 75, 83, 93, 99, 101, 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89,91, 95, 97, 99 or 101.

In one embodiment, the framework region is mutated so that the resultingframework region(s) have the amino acid sequence of the correspondinggermline gene. A mutation may be made in a framework region or constantdomain to increase the half-life of the anti-CD40 antibody. See, e.g.,PCT Publication No. WO 00/09560, herein incorporated by reference. Amutation in a framework region or constant domain also can be made toalter the immunogenicity of the antibody, to provide a site for covalentor non-covalent binding to another molecule, or to alter such propertiesas complement fixation, FcR binding and ADCC. According to theinvention, a single antibody may have mutations in any one or more ofthe framework regions, the constant domain and in the variable regions.

In some embodiments, there are from 1 to 18, including any number inbetween, amino acid mutations in either the V_(H) or V_(L) domains ofthe mutated anti-CD40 antibody compared to the anti-CD40 antibody priorto mutation. In any of the above, the mutations may occur in one or moreCDR regions. Further, any of the mutations can be conservative aminoacid substitutions. In some embodiments, there are no more than 5, 4, 3,2, or 1 amino acid changes in the constant domains.

Modified Antibodies

In another embodiment, a fusion antibody or immunoadhesin may be madethat comprises all or a portion of an anti-CD40 antibody of theinvention linked to another polypeptide. In a preferred embodiment, onlythe variable domains of the anti-CD40 antibody are linked to thepolypeptide. In another preferred embodiment, the V_(H) domain of ananti-CD40 antibody is linked to a first polypeptide, while the V_(L)domain of an anti-CD40 antibody is linked to a second polypeptide thatassociates with the first polypeptide in a manner such that the V_(H)and V_(L) domains can interact with one another to form an antibodybinding site. In another preferred embodiment, the V_(H) domain isseparated from the V_(L) domain by a linker such that the V_(H) andV_(L) domains can interact with one another (see below under SingleChain Antibodies). The V_(H)-linker-V_(L) antibody is then linked to thepolypeptide of interest. The fusion antibody is useful for directing apolypeptide to a CD40− expressing cell or tissue. The polypeptide may bea therapeutic agent, such as a toxin, growth factor or other regulatoryprotein, or may be a diagnostic agent, such as an enzyme that may beeasily visualized, such as horseradish peroxidase. In addition, fusionantibodies can be created in which two (or more) single-chain antibodiesare linked to one another. This is useful if one wants to create adivalent or polyvalent antibody on a single polypeptide chain, or if onewants to create a bispecific antibody.

To create a single chain antibody, (scFv) the V_(H)- and V_(L)-encodingDNA fragments are operatively linked to another fragment encoding aflexible linker, e.g., encoding the amino acid sequence (Gly₄-Ser)₃ (SEQID NO: 143), such that the V_(H) and V_(L) sequences can be expressed asa contiguous single-chain protein, with the V_(L) and V_(H) domainsjoined by the flexible linker. See, e.g., Bird et al., Science242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA85:5879-5883 (1988); McCafferty et al., Nature 348:552-554 (1990). Thesingle chain antibody may be monovalent, if only a single V_(H) andV_(L) are used, bivalent, if two V_(H) and V_(L) are used, orpolyvalent, if more than two V_(H) and V_(L) are used. Bispecific orpolyvalent antibodies may be generated that bind specifically to CD40and to another molecule.

In other embodiments, other modified antibodies may be prepared usinganti-CD40 antibody-encoding nucleic acid molecules. For instance, “Kappabodies” (Ill et al., Protein Eng. 10: 949-57 (1997)), “Minibodies”(Martin et al., EMBO J. 13: 5303-9 (1994)), “Diabodies” (Holliger etal., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)), or “Janusins”(Traunecker et al., EMBO J. 10:3655-3659 (1991) and Traunecker et al.,Int. J. Cancer (Suppl.) 7:51-52 (1992)) may be prepared using standardmolecular biological techniques following the teachings of thespecification.

Bispecific antibodies or antigen-binding fragments can be produced by avariety of methods including fusion of hybridomas or linking of Fab′fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990), Kostelny et al., J. Immunol. 148:1547-1553 (1992). Inaddition, bispecific antibodies may be formed as “diabodies” or“Janusins.” In some embodiments, the bispecific antibody binds to twodifferent epitopes of CD40. In some embodiments, the bispecific antibodyhas a first heavy chain and a first light chain from monoclonal antibody3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2,10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 22.1.1H-C109A, 23.5.1, 23.25.1,23.28.1, 23.28.1H-D16E, 23.28.1L-C92A, 23.29.1, 23.29.1L-R174K and24.2.1, and an additional antibody heavy chain and light chain. In someembodiments, the additional light chain and heavy chain also are fromone of the above-identified monoclonal antibodies, but are differentfrom the first heavy and light chains.

In some embodiments, the modified antibodies described above areprepared using one or more of the variable domains or CDR regions from ahuman anti-CD40 monoclonal antibody provided herein, from an amino acidsequence of said monoclonal antibody, or from a heavy chain or lightchain encoded by a nucleic acid sequence encoding said monoclonalantibody.

Derivatized and Labeled Antibodies

An anti-CD40 antibody or antigen-binding portion of the invention can bederivatized or linked to another molecule (e.g., another peptide orprotein). In general, the antibodies or portion thereof is derivatizedsuch that the CD40 binding is not affected adversely by thederivatization or labeling. Accordingly, the antibodies and antibodyportions of the invention are intended to include both intact andmodified forms of the human anti-CD40 antibodies described herein. Forexample, an antibody or antibody portion of the invention can befunctionally linked (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas another antibody (e.g., a bispecific antibody or a diabody), adetection agent, a cytotoxic agent, a pharmaceutical agent, and/or aprotein or peptide that can mediate associate of the antibody orantibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Another type of derivatized antibody is a labeled antibody. Usefuldetection agents with which an antibody or antigen-binding portion ofthe invention may be derivatized include fluorescent compounds,including fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthamidephosphors and the like. An antibody can also be labeled with enzymesthat are useful for detection, such as horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase andthe like. When an antibody is labeled with a detectable enzyme, it isdetected by adding additional reagents that the enzyme uses to produce areaction product that can be discerned. For example, when the agenthorseradish peroxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody can also be labeled with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Anantibody can also be labeled with a predetermined polypeptide epitoperecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, labels are attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

An anti-CD40 antibody can also be labeled with a radiolabeled aminoacid. The radiolabel can be used for both diagnostic and therapeuticpurposes. For instance, the radiolabel can be used to detectCD40-expressing tumors by x-ray or other diagnostic techniques. Further,the radiolabel can be used therapeutically as a toxin for cancerouscells or tumors. Examples of labels for polypeptides include, but arenot limited to, the following radioisotopes or radionuclides—³H, ¹⁴C,¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I.

An anti-CD40 antibody can also be derivatized with a chemical group suchas polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrategroup. These groups are useful to improve the biological characteristicsof the antibody, e.g., to increase serum half-life or to increase tissuebinding.

Pharmaceutical Compositions and Kits

The invention also relates to compositions comprising a human anti-CD40agonist antibody for the treatment of subjects in need ofimmunostimulation. Such compositions are useful to treat, prevent,reduce the frequency of or severity of infection, including viral andbacterial infection, for treating a hyperproliferative disorder,including cancerous and pre-cancerous conditions, for treating geneticimmunodeficiency conditions, such as hyper-IgM syndrome and for treatingprimary or combined immunodeficiency conditions, including conditionscharacterized by neutropenia, in a mammal, including humans. Subjectsfor treatment with agonist anti-CD40 antibody therapy include anysubject in need of immune enhancement, including but not limited to theelderly and individuals who are immunosuppressed, for example due tochemotherapy.

Hyperproliferative disorders that may be treated by an agonist anti-CD40antibody of the invention can involve any tissue or organ and includebut are not limited to brain, lung, squamous cell, bladder, gastric,pancreatic, breast, head, neck, liver, renal, ovarian, prostate,colorectal, esophageal, gynecological, nasopharynx, or thyroid cancers,melanomas, lymphomas, leukemias or multiple myelomas. In particular,human agonist anti-CD40 antibodies of the invention are useful to treatcarcinomas of the breast, prostate, colon and lung.

Treatment may involve administration of one or more agonist anti-CD40monoclonal antibodies of the invention, or antigen-binding fragmentsthereof, alone or with a pharmaceutically acceptable carrier. As usedherein, “pharmaceutically acceptable carrier” means any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Some examples of pharmaceutically acceptablecarriers are water, saline, phosphate buffered saline, dextrose,glycerol, ethanol and the like, as well as combinations thereof. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Additional examples of pharmaceutically acceptablesubstances are wetting agents or minor amounts of auxiliary substancessuch as wetting or emulsifying agents, preservatives or buffers, whichenhance the shelf life or effectiveness of the antibody.

Agonist anti-CD40 antibodies of the invention and compositionscomprising them, can be administered in combination with one or moreother therapeutic, diagnostic or prophylactic agents. Additionaltherapeutic agents include other anti-neoplastic, anti-tumor,anti-angiogenic or chemotherapeutic agents. Such additional agents maybe included in the same composition or administered separately. In someembodiments, one or more agonist anti-CD40 antibodies of the inventioncan be used as a vaccine or as adjuvants to a vaccine.

The compositions of this invention may be in a variety of forms, forexample, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Thepreferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans. The preferred mode ofadministration is parenteral (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular). In a preferred embodiment, the antibodyis administered by intravenous infusion or injection. In anotherpreferred embodiment, the antibody is administered by intramuscular orsubcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the anti-CD40 antibody in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

The antibodies of the present invention can be administered by a varietyof methods known in the art, although for many therapeutic applications,the preferred route/mode of administration is subcutaneous,intramuscular, or intravenous infusion. As will be appreciated by theskilled artisan, the route and/or mode of administration will varydepending upon the desired results.

In certain embodiments, the antibody compositions active compound may beprepared with a carrier that will protect the antibody against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems (J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978).

In certain embodiments, an anti-CD40 antibody of the invention can beorally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) can also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the anti-CD40 antibodies canbe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. To administer a compound of the inventionby other than parenteral administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation.

Additional active compounds also can be incorporated into thecompositions. In certain embodiments, an anti-CD40 antibody of theinvention is co-formulated with and/or co-administered with one or moreadditional therapeutic agents. These agents include, without limitation,antibodies that bind other targets (e.g., antibodies that bind one ormore growth factors or cytokines or their cell surface receptors, suchas anti-CTL4-antibody), antineoplastic agents, antitumor agents,chemotherapeutic agents, peptide analogues that activate CD40, solubleCD40L, one or more chemical agents that activates CD40, and/or otheragents known in the art that can enhance an immune response againsttumor cells, e.g., IFN-β1, IL-2, IL-8, IL-12, IL-15, IL-18, IL-23,IFN-γ, and GM-CSF. Such combination therapies may require lower dosagesof the anti-CD40 antibody as well as the co-administered agents, thusavoiding possible toxicities or complications associated with thevarious monotherapies.

Agonist anti-CD40 antibodies of the invention and compositionscomprising them also may be administered in combination with othertherapeutic regimens, in particular in combination with radiationtreatment.

The compositions of the invention may include a “therapeuticallyeffective amount” or a “prophylactically effective amount” of anantibody or antigen-binding portion of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the antibody or antibody portion mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody or antibody portion are outweighedby the therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus can be administered, several divided doses can be administeredover time or the dose can be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the anti-CD40 antibody or portion and the particulartherapeutic or prophylactic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an antibody for thetreatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.025 to 50 mg/kg, more preferably 0.1 to 50 mg/kg,more preferably 0.1-25, 0.1 to 10 or 0.1 to 3 mg/kg. It is to be notedthat dosage values may vary with the type and severity of the conditionto be alleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

Another aspect of the present invention provides kits comprising ananti-CD40 antibody or antibody portion of the invention or a compositioncomprising such an antibody. A kit may include, in addition to theantibody or composition, diagnostic or therapeutic agents. A kit canalso include instructions for use in a diagnostic or therapeutic method.In a preferred embodiment, the kit includes the antibody or acomposition comprising it and a diagnostic agent that can be used in amethod described below. In another preferred embodiment, the kitincludes the antibody or a composition comprising it and one or moretherapeutic agents that can be used in a method described below.

This invention also relates to compositions for inhibiting abnormal cellgrowth in a mammal comprising an amount of an antibody of the inventionin combination with an amount of a chemotherapeutic, wherein the amountsof the compound, salt, solvate, or prodrug, and of the chemotherapeuticare together effective in inhibiting abnormal cell growth. Manychemotherapeutics are presently known in the art. In some embodiments,the chemotherapeutic is selected from the group consisting of mitoticinhibitors, alkylating agents, anti-metabolites, intercalatingantibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes,topoisomerase inhibitors, biological response modifiers, anti-hormones,e.g. anti-androgens, and anti-angiogenesis agents.

Anti-angiogenic agents, such as MMP-2 (matrix-metalloproteinase 2)inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, and COX-II(cyclooxygenase II) inhibitors, can be used in conjunction with ananti-CD40 antibody of the invention. Examples of useful COX-IIinhibitors include CELEBREX™ (alecoxib), valdecoxib, and rofecoxib.Examples of useful matrix metalloproteinase inhibitors are described inWO 96/33172 (published Oct. 24, 1996), WO 96/27583 (published Mar. 7,1996), European Patent Application No. 97304971.1 (filed Jul. 8, 1997),European Patent Application No. 99308617.2 (filed Oct. 29, 1999), WO98/07697 (published Feb. 26, 1998), WO 98/03516 (published Jan. 29,1998), WO 98/34918 (published Aug. 13, 1998), WO 98/34915 (publishedAug. 13, 1998), WO 98/33768 (published Aug. 6, 1998), WO 98/30566(published Jul. 16, 1998), European Patent Publication 606,046(published Jul. 13, 1994), European Patent Publication 931,788(published Jul. 28, 1999), WO 90/05719 (published May 31, 1990), WO99/52910 (published Oct. 21, 1999), WO 99/52889 (published Oct. 21,1999), WO 99/29667 (published Jun. 17, 1999), PCT InternationalApplication No. PCT/IB98/01113 (filed Jul. 21, 1998), European PatentApplication No. 99302232.1 (filed Mar. 25, 1999), Great Britain patentapplication number 9912961.1 (filed Jun. 3, 1999), U.S. ProvisionalApplication No. 60/148,464 (filed Aug. 12, 1999), U.S. Pat. No.5,863,949 (issued Jan. 26, 1999), U.S. Pat. No. 5,861,510 (issued Jan.19, 1999), and European Patent Publication 780,386 (published Jun. 25,1997), all of which are incorporated herein in their entireties byreference. Preferred MMP inhibitors are those that do not demonstratearthralgia. More preferred, are those that selectively inhibit MMP-2and/or MMP-9 relative to the other matrix-metalloproteinases (i.e.MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12,and MMP-13). Some specific examples of MMP inhibitors useful in thepresent invention are AG-3340, RO 32-3555, RS 13-0830, and the compoundsrecited in the following list:3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclopentyl)-amino]-propionicacid;3-exo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(2-chloro-4-fluoro-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;4-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclobutyl)-amino]-propionicacid;4-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide; (R)3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(4-fluoro-2-methyl-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-1-methyl-ethyl)-amino]-propionicacid;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro-pyran-4-yl)-amino]-propionic acid;3-exo-3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide;3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; and (R)3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-furan-3-carboxylicacid hydroxyamide; and pharmaceutically acceptable salts and solvates ofsaid compounds.

A compound of the invention can also be used with signal transductioninhibitors, such as agents that can inhibit EGF-R (epidermal growthfactor receptor) responses, such as EGF-R antibodies, EGF antibodies,and molecules that are EGF-R inhibitors; VEGF (vascular endothelialgrowth factor) inhibitors, such as VEGF receptors and molecules that caninhibit VEGF; and erbB2 receptor inhibitors, such as organic moleculesor antibodies that bind to the erbB2 receptor, for example, HERCEPTIN®(Genentech, Inc.). EGF-R inhibitors are described in, for example in WO95/19970 (published Jul. 27, 1995), WO 98/14451 (published Apr. 9,1998), WO 98/02434 (published Jan. 22, 1998), and U.S. Pat. No.5,747,498 (issued May 5, 1998), and such substances can be used in thepresent invention as described herein. EGFR-inhibiting agents include,but are not limited to, the monoclonal antibodies C225 and anti-EGFR22Mab (ImClone Systems Incorporated), ABX-EGF (Abgenix/Cell Genesys),EMD-7200 (Merck KgaA), EMD-5590 (Merck KgaA), MDX-447/H-477 (MedarexInc. and Merck KgaA), and the compounds ZD-1834, ZD-1838 and ZD-1839(AstraZeneca), PKI-166 (Novartis), PKI-166/CGP-75166 (Novartis), PTK 787(Novartis), CP 701 (Cephalon), leflunomide (Pharmacia/Sugen), CI-1033(Warner Lambert Parke Davis), CI-1033/PD 183,805 (Warner Lambert ParkeDavis), CL-387,785 (Wyeth-Ayerst), BBR-1611 (Boehringer MannheimGmbH/Roche), Naamidine A (Bristol Myers Squibb), RC-3940-II (Pharmacia),BIBX-1382 (Boehringer Ingelheim), OLX-103 (Merck & Co.), VRCTC-310(Ventech Research), EGF fusion toxin (Seragen Inc.), DAB-389(Seragen/Lilgand), ZM-252808 (Imperial Cancer Research Fund), RG-50864(INSERM), LFM-A12 (Parker Hughes Cancer Center), WH1-P97 (Parker HughesCancer Center), GW-282974 (Glaxo), KT-8391 (Kyowa Hakko) and EGF-RVaccine (York Medical/Centro de Immunologia Molecular (CIM)). These andother EGF-R-inhibiting agents can be used in the present invention.

VEGF inhibitors, for example SU-5416 and SU-6668 (Sugen Inc.), SH-268(Schering), and NX-1838 (NeXstar) can also be combined with the compoundof the present invention. VEGF inhibitors are described in, for examplein WO 99/24440 (published May 20, 1999), PCT International ApplicationPCT/EB99/00797 (filed May 3, 1999), in WO 95/21613 (published Aug. 17,1995), WO 99/61422 (published Dec. 2, 1999), U.S. Pat. No. 5,834,504(issued Nov. 10, 1998), WO 98/50356 (published Nov. 12, 1998), U.S. Pat.No. 5,883,113 (issued Mar. 16, 1999), U.S. Pat. No. 5,886,020 (issuedMar. 23, 1999), U.S. Pat. No. 5,792,783 (issued Aug. 11, 1998), WO99/10349 (published Mar. 4, 1999), WO 97/32856 (published Sep. 12,1997), WO 97/22596 (published Jun. 26, 1997), WO 98/54093 (publishedDec. 3, 1998), WO 98/02438 (published Jan. 22, 1998), WO 99/16755(published Apr. 8, 1999), and WO 98/02437 (published Jan. 22, 1998), allof which are incorporated herein in their entireties by reference. Otherexamples of some specific VEGF inhibitors useful in the presentinvention are IM862 (Cytran Inc.); anti-VEGF monoclonal antibody ofGenentech, Inc.; and angiozyme, a synthetic ribozyme from Ribozyme andChiron. These and other VEGF inhibitors can be used in the presentinvention as described herein. ErbB2 receptor inhibitors, such asGW-282974 (Glaxo Wellcome plc), and the monoclonal antibodies AR-209(Aronex Pharmaceuticals Inc.) and 2B-1 (Chiron), can furthermore becombined with the compound of the invention, for example those indicatedin WO 98/02434 (published Jan. 22, 1998), WO 99/35146 (published Jul.15, 1999), WO 99/35132 (published Jul. 15, 1999), WO 98/02437 (publishedJan. 22, 1998), WO 97/13760 (published Apr. 17, 1997), WO 95/19970(published Jul. 27, 1995), U.S. Pat. No. 5,587,458 (issued Dec. 24,1996), and U.S. Pat. No. 5,877,305 (issued Mar. 2, 1999), which are allhereby incorporated herein in their entireties by reference. ErbB2receptor inhibitors useful in the present invention are also describedin U.S. Provisional Application No. 60/117,341, filed Jan. 27, 1999, andin U.S. Provisional Application No. 60/117,346, filed Jan. 27, 1999,both of which are incorporated in their entireties herein by reference.The erbB2 receptor inhibitor compounds and substance described in theaforementioned PCT applications, U.S. patents, and U.S. provisionalapplications, as well as other compounds and substances that inhibit theerbB2 receptor, can be used with the compound of the present inventionin accordance with the present invention.

Anti-survival agents include anti-IGF-IR antibodies and anti-integrinagents, such as anti-integrin antibodies.

Diagnostic Methods of Use

In another aspect, the invention provides diagnostic methods. Theanti-CD40 antibodies can be used to detect CD40 in a biological samplein vitro or in vivo. In one embodiment, the invention provides a methodfor diagnosing the presence or location of an CD40-expressing tumor in asubject in need thereof, comprising the steps of injecting the antibodyinto the subject, determining the expression of CD40 in the subject bylocalizing where the antibody has bound, comparing the expression in thesubject with that of a normal reference subject or standard, anddiagnosing the presence or location of the tumor.

The anti-CD40 antibodies can be used in a conventional immunoassay,including, without limitation, an ELISA, an RIA, FACS, tissueimmunohistochemistry, Western blot or immunoprecipitation. The anti-CD40antibodies of the invention can be used to detect CD40 from humans. Inanother embodiment, the anti-CD40 antibodies can be used to detect CD40from Old World primates such as cynomolgus and rhesus monkeys,chimpanzees and apes. The invention provides a method for detecting CD40in a biological sample comprising contacting a biological sample with ananti-CD40 antibody of the invention and detecting the bound antibody. Inone embodiment, the anti-CD40 antibody is directly labeled with adetectable label. In another embodiment, the anti-CD40 antibody (thefirst antibody) is unlabeled and a second antibody or other moleculethat can bind the anti-CD40 antibody is labeled. As is well known to oneof skill in the art, a second antibody is chosen that is able tospecifically bind the particular species and class of the firstantibody. For example, if the anti-CD40 antibody is a human IgG, thenthe secondary antibody could be an anti-human-IgG. Other molecules thatcan bind to antibodies include, without limitation, Protein A andProtein G, both of which are available commercially, e.g., from PierceChemical Co.

Suitable labels for the antibody or secondary antibody have beendisclosed supra, and include various enzymes, prosthetic groups,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In other embodiments, CD40 can be assayed in a biological sample by acompetition immunoassay utilizing CD40 standards labeled with adetectable substance and an unlabeled anti-CD40 antibody. In this assay,the biological sample, the labeled CD40 standards and the anti-CD40antibody are combined and the amount of labeled CD40 standard bound tothe unlabeled antibody is determined. The amount of CD40 in thebiological sample is inversely proportional to the amount of labeledCD40 standard bound to the anti-CD40 antibody.

One can use the immunoassays disclosed above for a number of purposes.For example, the anti-CD40 antibodies can be used to detect CD40 incells in cell culture. In a preferred embodiment, the anti-CD40antibodies are used to determine the amount of CD40 on the surface ofcells that have been treated with various compounds. This method can beused to identify compounds that are useful to activate or inhibit CD40.According to this method, one sample of cells is treated with a testcompound for a period of time while another sample is left untreated. Ifthe total level of CD40 is to be measured, the cells are lysed and thetotal CD40 level is measured using one of the immunoassays describedabove. The total level of CD40 in the treated versus the untreated cellsis compared to determine the effect of the test compound.

A preferred immunoassay for measuring total CD40 levels is an ELISA orWestern blot. If the cell surface level of CD40 is to be measured, thecells are not lysed, and the cell surface levels of CD40 are measuredusing one of the immunoassays described above. A preferred immunoassayfor determining cell surface levels of CD40 includes the steps oflabeling the cell surface proteins with a detectable label, such asbiotin or ¹²⁵I, immunoprecipitating the CD40 with an anti-CD40 antibodyand then detecting the labeled CD40. Another preferred immunoassay fordetermining the localization of CD40, e.g., cell surface levels, is byusing immunohistochemistry. Methods such as ELISA, RIA, Western blot,immunohistochemistry, cell surface labeling of integral membraneproteins and immunoprecipitation are well known in the art. See, e.g.,Harlow and Lane, supra. In addition, the immunoassays can be scaled upfor high throughput screening in order to test a large number ofcompounds for either activation or inhibition of CD40.

The anti-CD40 antibodies of the invention can also be used to determinethe levels of CD40 in a tissue or in cells derived from the tissue. Insome embodiments, the tissue is a diseased tissue. In some embodiments,the tissue is a tumor or a biopsy thereof. In some embodiments of themethod, a tissue or a biopsy thereof is excised from a patient. Thetissue or biopsy is then used in an immunoassay to determine, e.g.,total CD40 levels, cell surface levels of CD40 or localization of CD40by the methods discussed above.

The above-described diagnostic method can be used to determine whether atumor expresses high levels of CD40, which could be indicative that thetumor is a target for treatment with anti-CD40 antibody. Further, thesame method can also be used to monitor the effect of the treatment withanti-CD40 antibody by detecting cell death in the tumor. The diagnosticmethod can also be used to determine whether a tissue or cell expressesinsufficient levels of CD40 or activated CD40, and thus is a candidatefor treatment with activating anti-CD40 antibodies, CD40L and/or othertherapeutic agents for increasing CD40 levels or activity.

The antibodies of the present invention can also be used in vivo toidentify tissues and organs that express CD40. In some embodiments, theanti-CD40 antibodies are used to identify CD40-expressing tumors. Oneadvantage of using the human anti-CD40 antibodies of the presentinvention is that they may safely be used in vivo without eliciting animmune response to the antibody upon administration, unlike antibodiesof non-human origin or with humanized antibodies.

The method comprises the steps of administering a detectably labeled ananti-CD40 antibody or a composition comprising them to a patient in needof such a diagnostic test and subjecting the patient to imaging analysisto determine the location of the CD40-expressing tissues. Imaginganalysis is well known in the medical art, and includes, withoutlimitation, x-ray analysis, magnetic resonance imaging (MRI) or computedtomography (CE). The antibody can be labeled with any agent suitable forin vivo imaging, for example a contrast agent, such as barium, which canbe used for x-ray analysis, or a magnetic contrast agent, such as agadolinium chelate, which can be used for MRI or CE. Other labelingagents include, without limitation, radioisotopes, such as ⁹⁹Tc. Inanother embodiment, the anti-CD40 antibody will be unlabeled and will beimaged by administering a second antibody or other molecule that isdetectable and that can bind the anti-CD40 antibody. In embodiment, abiopsy is obtained from the patient to determine whether the tissue ofinterest expresses CD40.

Therapeutic Methods of use

In another aspect, invention provides therapeutic methods of using ananti-CD40 antibody of the invention.

A human agonist anti-CD40 antibody of the invention can be administeredto a human or to a non-human mammal that expresses a cross-reactingCD40. The antibody can be administered to such a non-human mammal (i.e.,a primate, cynomolgus or rhesus monkey) for veterinary purposes or as ananimal model of human disease. Such animal models are useful forevaluating the therapeutic efficacy of antibodies of this invention.

In some embodiments, the anti-CD40 antibody is administered to a subjectwho suffers from primary and/or combined immunodeficiencies, includingCD40− dependent immunodeficiency with Hyper-IgM syndrome, CommonVariable Immunodeficiency, Bruton's Agammaglobulinemia, IgG subclassdeficiencies, and X-linked SCID (common gamma chain mutations). In someembodiments, the anti-CD40 antibody is administered to treat a subjectwho is immunosuppressed, for example due to chemotherapy, or has animmune-debilitating disease, including any acquired immune deficiencydisease, such as HIV. In some embodiments, the anti-CD40 antibody isadministered to enhance the immunity of an elderly subject. In someembodiments, the anti-CD40 antibody is administered to treat a subjectwho has a bacterial, viral, fungal or parasitic infection. In someembodiments, a human agonist anti-CD40 antibody of the invention may beadministered prophylactically to a subject who, because of age, illnessor general poor health is susceptible to infection to prevent or toreduce the number or severity of infections.

In some embodiments, the anti-CD40 antibody is administered to a subjectwho has a hyperproliferative disorder.

In some embodiments, the anti-CD40 antibody is administered to treat asubject who has a tumor. In some embodiments, the tumor is CD40positive. In some embodiments, the tumor is a CD40 negative. The tumorcan be a solid tumor or a non-solid tumor such as lymphoma. In someembodiments, an anti-CD40 antibody is administered to a patient who hasa tumor that is cancerous. In some embodiments, the antibody inhibitscancer cell proliferation, inhibits or prevents an increase in tumorweight or volume, and/or causes a decrease in tumor weight or volume.

Patients that can be treated with anti-CD40 antibodies or antibodyportions of the invention include, but are not limited to, patients thathave been diagnosed as having brain cancer, lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head and neck, cutaneousor intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colorectalcancer, colon cancer, breast cancer, gynecologic tumors (e.g., uterinesarcomas, carcinoma of the fallopian tubes, carcinoma of theendometrium, carcinoma of the cervix, carcinoma of the vagina orcarcinoma of the vulva), cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system (e.g., cancer of the thyroid,parathyroid or adrenal glands), sarcomas of soft tissues, leukemia,myeloma, multiple myeloma, cancer of the urethra, cancer of the penis,prostate cancer, chronic or acute leukemia, solid tumors of childhood,Hodgkin's disease, lymphocytic lymphomas, non-Hodgkin lymphoma, cancerof the bladder, liver cancer, renal cancer, cancer of the kidney orureter (e.g., renal cell carcinoma, carcinoma of the renal pelvis), orneoplasms of the central nervous system (e.g., primary CNS lymphoma,spinal axis tumors, brain stem gliomas or pituitary adenomas), glioma orfibrosarcoma.

The antibody may be administered from three times daily to once everysix months, and preferably may be administered via an oral, mucosal,buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular,parenteral, intratumor, transdermal or topical route. The antibody canalso be administered continuously via a minipump. The antibody generallywill be administered for as long as the tumor is present provided thatthe antibody causes the tumor or cancer to stop growing or to decreasein weight or volume. The dosage of antibody generally will be in therange of 0.025 to 50 mg/kg, more preferably 0.1 to 50 mg/kg, morepreferably 0.1-20 mg/kg, 0.1-10 mg/kg, 0.1-5 mg/kg or even morepreferable 0.1-2 mg/kg. The antibody can also be administeredprophylactically.

In some embodiments, the anti-CD40 antibody is administered as part of atherapeutic regimen that includes one or more additional antineoplasticdrugs or molecules to a patient who has a hyperproliferative disorder,such as cancer or a tumor. Exemplary antitumor agents include, but arenot limited to, mitotic inhibitors, alkylating agents, anti-metabolites,intercalating agents, growth factor inhibitors, cell cycle inhibitors,enzymes, topoisomerase inhibitors, biological response modifiers,anti-hormones, kinase inhibitors, matrix metalloprotease inhibitors,genetic therapeutics and anti-androgens. In more preferred embodiments,the anti-CD40 antibody is administered with an antineoplastic agent,such as adriamycin or taxol. In some preferred embodiments, theanti-CD40 therapy is performed along with radiotherapy, chemotherapy,photodynamic therapy, surgery or other immunotherapy. In someembodiments, the anti-CD40 antibody is administered with one or moreadditional antibodies. For example, the anti-CD40 antibody can beadministered with antibodies that are known to inhibit tumor or cancercell proliferation. Such antibodies include, but are not limited to, anantibody that inhibits CTLA4, erbB2 receptor, EGF-R, IGF-1R, CD20 orVEGF.

In some embodiments, the anti-CD40 antibody is labeled with aradiolabel, an immunotoxin or a toxin, or is a fusion protein comprisinga toxic peptide. The anti-CD40 antibody or anti-CD40 antibody fusionprotein directs the radiolabel, immunotoxin, toxin or toxic peptide tothe tumor or cancer cell. In a preferred embodiment, the radiolabel,immunotoxin, toxin or toxic peptide is internalized by the tumor orcancer cell after the anti-CD40 antibody binds to the CD40 on thesurface of the cell.

In another aspect, the anti-CD40 antibody can be used therapeutically toinduce apoptosis of specific cells in a patient. In many cases, thecells targeted for apoptosis are cancerous or tumor cells. Thus, theinvention provides a method of inducing apoptosis by administering ananti-CD40 antibody to a patient in need thereof.

In another aspect, the invention provides a method of administering anactivating anti-CD40 antibody to a patient to increase CD40 activity. Ananti-CD40 antibody is administered with one or more other factors thatincrease CD40 activity. Such factors include CD40L, and/or analogues ofCD40L that activate CD40.

In some embodiments, the anti-CD40 antibody is administered with one ormore additional immune enhancing agents, including, without limitationIFN-β1, IL-2, IL-8, IL-12, IL-15, IL-18, IL-23, IFN-γ, and GM-CSF.

In some embodiments, a human agonist anti-CD40 antibody of the inventionis used as an adjuvant to enhance the efficacy of a vaccine. When usedin this way, the anti-CD-40 antibody activates CD40 on antigenpresenting cells, including B cells, dendritic cells and monocytes aswell as enhancing the production of immunomodulatory molecules, such ascytokines and chemokines. The immunostimulatory effect of the antibodyenhances the immune response of the vaccinated subject to the vaccineantigen.

In another aspect, the invention provides a method for generating adendritic cell vaccine for cancer or for dendritic cell immunotherapy.According to the method dendritic cells from a cancer patient arecultured for 1-5 days with tumor lysate or homogenate, tumor cellskilled by irradiation or other means, or tumor specific antigens (e.g.,peptides, idiotypes) and 1-10 μg/ml of an anti-CD40 antibody. The tumorantigen-pulsed dendritic cells are re-injected into the patient tostimulate anti-tumor immune responses, particularly anti-tumor CTLresponses. Monocyte-derived dendritic cells for use in the method can beobtained from a peripheral blood sample by culture in IL-4 and GM-CSF.Dendritic cells also can be derived from the bone marrow of a patient bymagnetic purification or sorting of CD34 positive cells, followed byculture in IL-4 and GM-CSF.

Gene Therapy

The nucleic acid molecules of the instant invention can be administeredto a patient in need thereof via gene therapy. The therapy may be eitherin vivo or ex vivo. In a preferred embodiment, nucleic acid moleculesencoding both a heavy chain and a light chain are administered to apatient. In a more preferred embodiment, the nucleic acid molecules areadministered such that they are stably integrated into chromosomes of Bcells because these cells are specialized for producing antibodies. In apreferred embodiment, precursor B cells are transfected or infected exvivo and re-transplanted into a patient in need thereof. In anotherembodiment, precursor B cells or other cells are infected in vivo usinga virus known to infect the cell type of interest. Typical vectors usedfor gene therapy include liposomes, plasmids and viral vectors.Exemplary viral vectors are retroviruses, adenoviruses andadeno-associated viruses. After infection either in vivo or ex vivo,levels of antibody expression can be monitored by taking a sample fromthe treated patient and using any immunoassay known in the art ordiscussed herein.

In a preferred embodiment, the gene therapy method comprises the stepsof administering an isolated nucleic acid molecule encoding the heavychain or an antigen-binding portion thereof of an anti-CD40 antibody andexpressing the nucleic acid molecule. In another embodiment, the genetherapy method comprises the steps of administering an isolated nucleicacid molecule encoding the light chain or an antigen-binding portionthereof of an anti-CD40 antibody and expressing the nucleic acidmolecule. In a more preferred method, the gene therapy method comprisesthe steps of administering of an isolated nucleic acid molecule encodingthe heavy chain or an antigen-binding portion thereof and an isolatednucleic acid molecule encoding the light chain or the antigen-bindingportion thereof of an anti-CD40 antibody of the invention and expressingthe nucleic acid molecules. The gene therapy method may also comprisethe step of administering another anti-cancer agent, such as taxol oradriamycin.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly and are not to be construed as limiting the scope of the inventionin any manner.

EXAMPLE I Generation of Hybridomas Producing Anti-CD40 Antibody

Antibodies of the invention were prepared, selected, and assayed asfollows:

Immunization and Hybridoma Generation

We immunized eight to ten week old XenoMice™ intraperitoneally or intheir hind footpads with either a CD40-IgG fusion protein (10μg/dose/mouse) or with 300.19-CD40 cells which is a transfected cellline that express human CD40 on its plasma membrane (10×10⁶cells/dose/mouse). We repeated this dose five to seven times over athree to eight week period. Four days before fusion, we gave the mice afinal injection of the extracellular domain of human CD40 in PBS. Wefused the spleen and lymph node lymphocytes from immunized mice with thenon-secretory myeloma P3-X63-Ag8.653 cell line, and subjected the fusedcells to HAT selection as previously described (Galfre and Milstein,Methods Enzymol. 73:3-46, 1981). We recovered a panel of hybridomas allsecreting CD40 specific human IgG2κ antibodies. We selected elevenhybridomas for further study and designated them 3.1.1, 7.1.2, 10.8.3,15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.29.1 and 24.2.1.

We deposited hybridomas 3.1.1, 7.1.2, 10.8.3, 15.1.1 and 21.4.1 in theAmerican Type Culture Collection (ATCC) in accordance with the BudapestTreaty, 10801 University Boulevard, Manassas, Va. 20110-2209, on Aug. 6,2001. We deposited hybridomas 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1,23.29.1 and 24.2.1 in the ATCC on Jul. 16, 2002. The hybridomas havebeen assigned the following deposit numbers:

Hybridoma Deposit No.  3.1.1 (LN 15848) PTA-3600  7.1.2 (LN 15849)PTA-3601  10.8.3 (LN 15850) PTA-3602  15.1.1 (LN 15851) PTA-3603  21.4.1(LN 15853) PTA-3605  21.2.1 (LN 15874) PTA-4549  22.1.1 (LN 15875)PTA-4550  23.5.1 (LN 15855) PTA-4548 23.25.1 (LN 15876) PTA-4551 23.28.1(LN 15877) PTA-4552 23.29.1 (LN 15878) PTA-4553  24.2.1 (LN 15879)PTA-4554

EXAMPLE II Sequences of Anti-CD40-Antibodies Prepared in Accordance withthe Invention

To analyze the structure of antibodies produced in accordance with theinvention, we cloned nucleic acids encoding heavy and light chainfragments from hybridomas producing anti-CD40 monoclonal antibodies.Cloning and sequencing was accomplished as follows.

We isolated Poly(A)⁺ mRNA from approximately 2×10⁵ hybridoma cellsderived from XenoMouse™ mice immunized with human CD40 as described inExample I using a Fast-Track kit (Invitrogen). We followed by PCR thegeneration of random primed cDNA. We used human V_(H) or human Vicfamily specific variable region primers (Marks et al., “Oligonucleotideprimers for polymerase chain reaction amplification of humanimmunoglobulin variable genes and design of family-specificoligonucleotide probes.” Eur. J. Immunol. 21:985-991 (1991)) or auniversal human V_(H) primer, MG-30, CAGGTGCAGCTGGAGCAGTCIGG (SEQ ID NO:118), in conjunction with primers specific for the human Cj2 constantregion, MG-40d, 5′-GCTGAGGGAGTAGAGTCCTGAGGA-3′ (SEQ ID NO: 119) or CKconstant region (hκP2; as previously described in Green et al., 1994).We obtained nucleic acid molecules encoding human heavy and kappa lightchain transcripts from the anti-CD40 producing hybridomas by directsequencing of PCR products generated from poly(A⁺) RNA using the primersdescribed above. We also cloned PCR products into pCRII using a TAcloning kit (Invitrogen) and sequenced both strands using Prismdye-terminator sequencing kits and an ABI 377 sequencing machine. Weanalyzed all sequences by alignments to the “V BASE sequence directory”(Tomlinson et al., MRC Centre for Protein Engineering, Cambridge, UK)using MacVector and Geneworks software programs.

Further, we subjected monoclonal antibodies 3.1.1, 7.1.2, 10.8.3,15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.28.1, 23.29.1 and 24.2.1 tofull length DNA cloning and sequencing. For such sequencing, we isolatedRNA from approximately 4×10⁶ hybridoma cells using QIAGEN RNeasy® RNAisolation kit (QIAGEN). We reverse transcribed the mRNA usingoligo-dT(18) (SEQ ID NO: 144) and the Advantage® RT/PCR kit (Clontech).We used V Base to design forward amplification primers that includedrestriction sites, optimal Kozak sequence, the ATG start site and partof the signal sequence of the heavy chain. Table 1 lists the forwardamplification primers used to sequence the antibody clones.

TABLE 1 Clone Forward Primer Heavy Chain 3.1.15′-TATCTAAGCTTCTAGACTCGACCGCCACCATGGAGTTT GGGCTGAGCTG-3′ (SEQ ID NO:120) 7.1.2 5′-TATCTAAGCTTCTAGACTCGACCGCCACCATGGAGTTT GGGCTGAGCTG-3′ (SEQID NO: 121) 10.8.3 5′-TATCTAAGCTTCTAGACTCGAGCGCCACCATGAAACACCTGTGGTTCTTCC-3′ (SEQ ID NO: 122) 15.1.15′-TATCTAAGCTTCTAGACTCGAGCGCCACCATGAAACAT CTGTGGTTCTTCC 3′ (SEQ ID NO:123) 21.4.1 5′-TATCTAAGCTTCTAGACTCGAGCGCCACCATGGACTGGACCTGGAGGATCC-3′ (SEQ ID NO: 124) 21.2.15′-TATCTAAGCTTCTAGACTCGACCGCCACCATGGA GTTTGGGCTGAGCTG-3′ (SEQ ID NO:128)22.1.1 5′-TATCTAAGCTTCTAGACTCGACCGCCACCATGGAG TTTGGGCTGAGCTG-3′ (SEQ IDNO:129) 23.5.1 5′-TATCTAAGCTTCTAGACTCGACCGCCACCATGGAGTTTGGGCTGAGCTG-3′ (SEQ ID NO:130) 23.28.15′-TATCTAAGCTTCTAGACTCGAGCGCCACCATGAAA CATCTGTGGTTCTTCC-3′ (SEQ IDNO:131) 23.29.1 5′-TATCTAAGCTTCTAGACTCGACCGCCACCATGGAGTTTGGGCTGAGCTG-3′ (SEQ ID NO:132) 24.2.15′-TATCTAAGCTTCTAGACTCGAGCGCCACCATGAA ACATCTGTGGTTCTTCC-3′ (SEQ IDNO:133)We used the same method to design a primer to include the 3′ codingsequences, the stop codon of the IgG2 constant region,(5′-TTCTCTGATCAGAATTCC TATCATTTACCCGGAGACAGGGAGAG-3′) (SEQ ID NO:125)and restriction sites.

We also used the same method to design a primer around the ATG startsite of the kappa chain: (5′-CTTCAAGCTTACCCGGGCCACCATGAGGCTCCCTGCTCAGC-3′) (SEQ ID NO:126). An optimal Kozak sequence (CCGCCACC) wasadded 5′ to the ATG start site. This primer was used to PCR clone thelight chains of following antibody clones: 3.1.1, 7.1.2, 10.8.3, 15.1.1,21.4.1, 21.2.1, 22.1.1, 23.5.1 and 23.29.1. We used a second forwardprimer 5′-TCTTC AAGCTTGCCCGGGCCCGCCACCATGGAAACCCCAGCGCAG-3′ (SEQ ID NO.134) to clone the light chains of clones 23.28.1 and 24.2.1. We alsoused the same method to design a primer around the stop codon of thekappa constant region(5′-TTCTTTGATCAGAATTCTCACTAACACTCTCCCCTGTTGAAGC-3′) (SEQ ID NO: 127). Weused the primer pairs to amplify the cDNAs using Advantage® HighFidelity PCR Kit (Clontech). We obtained the sequence of the PCR productby direct sequencing using standard techniques (e.g., primer walking)using dye-terminator sequencing kits and an ABI sequencing machine. Wecloned the PCR product into a mammalian expression vector and wesequenced clones to confirm somatic mutations. For each clone, weverified the sequence on both strands in at least three reactions.

Gene Utilization Analysis

Table 2 sets forth the gene utilization evidenced by selected hybridomaclones of antibodies in accordance with the invention:

TABLE 2 Heavy and Light Chain Gene Utilization Heavy Chain Kappa LightChain Clone VH D JH VK JK 3.1.1 (3-30+) D4+ JH6 A3/A19 JK1 DP-49 DIR3(DPK-15) 7.1.2 (3-30+) DIR5+ JH6 A3/A19 JK1 DP-49 D1-26 (DPK-15) 10.8.3(4.35) DIR3 JH6 L5 JK4 VIV-4 (DP5) 15.1.1 (4-59) D4-23 JH4 A3/A19 JK2DP-71 (DPK-15) 21.4.1 (1-02) DLR1 JH4 L5 JK4 DP-75 (DP5) 21.2.1 (3-30+)DIR3+ JH4 A3/A19 JK3 DP-49 D6-19 (DPK-15) 22.1.1 (3-30+) D1-1 JH6 A3/A19JK1 DP-49 (DPK-15) 23.5.1 (3-30+) D4-17 JH6 A3/A19 JK1 DP-49 (DPK-15)23.28.1 (4-59) DIR1+ JH5 A27 JK3 DP-71 D4-17 (DPK-22) 23.29.1 (3-30.3)D4-17 JH6 A3/A19 JK1 DP-46 (DPK-15) 24.2.1 (4-59) DIR1+ JH5 A27 JK3DP-71 D4-17 (DPK-22)Sequence And Mutation Analysis

As will be appreciated, gene utilization analysis provides only alimited overview of antibody structure. As the B-cells in XenoMouse™animals stochastically generate V-D-J heavy or V-J kappa light chaintranscripts, there are a number of secondary processes that occur,including, without limitation, somatic hypermutation, deletions,N-additions, and CDR3 extensions. See, for example, Mendez et al.,Nature Genetics 15:146-156 (1997) and International Patent PublicationWO 98/24893. Accordingly, to further examine antibody structure, wegenerated predicted amino acid sequences of the antibodies from thecDNAs obtained from the clones. Table A provides the sequenceidentifiers for each of the nucleotide and predicted amino acidsequences of the sequenced antibodies.

Tables 3-7 provide the nucleotide and predicted amino acid sequences ofthe heavy and kappa light chains of antibodies 3.1.1 (Table 3), 7.1.2(Table 4), 10.8.3 (Table 5), 15.1.1 (Table 6) and 21.4.1 (Table 7).

Tables 8-13 provide the nucleotide and predicted amino acid sequences ofthe variable domain of the heavy chain and kappa light chain ofantibodies 21.2.1 (Table 8), 22.1.1 (Table 9), 23.5.1 (Table 10),23.28.1 (Table 11), 23.29.1 (Table 12) and 24.2.1 (Table 13).

The DNA sequence from the full-length sequencing of monoclonal antibody23.28.1 differs from DNA sequences obtained from sequencing the V_(H)region of the initial PCR product by one base pair (C to G), resultingin a change of residue 16 of the natural heavy chain from D to E.

Tables 14-19 provide the nucleotide and predicted amino acid sequencesof the heavy and kappa light chains of antibodies 21.2.1 (Table 14),22.1.1 (Table 15), 23.5.1 (Table 16), 23.28.1 (Table 17), 23.29.1 (Table18) and 24.2.1 (Table 19). In the Tables, the signal peptide sequence(or the bases encoding the same) are underlined.

We generated two mutated antibodies, 22.1.1 and 23.28.1. The heavy chainof antibody 22.1.1 was mutated to change a cysteine residue at position109 to an alanine residue. We designated the mutated clone22.1.1H-C₀₁₉A. The light chain of antibody 23.28.1 at position 92 wasmutated also to change a cysteine residue to an alanine residue. Wedesignated the mutated clone 23.28.1L-C92A.

Mutagenesis of specific residues was carried out by designing primersand using the Quikchange® Site-Directed Mutagenesis Kit from Stratagene,according to the manufacturer's instructions. Mutations were confirmedby automated sequencing, and mutagenized inserts were subcloned intoexpression vectors.

Table 20 provides the nucleotide and amino acid sequences of the mutatedheavy chain of antibody 22.1.1H-C109A. Table 21 provides the nucleotideand amino acid sequences of the mutated light chain of antibody 23.28.1.The mutated DNA codons are shown in italics. The mutated amino acidresidue is in bold.

TABLE 3 DNA and protein sequences of antibody 3.1.1 DESCRIPTION:SEQUENCE (signal sequence underlined): Heavy ChainATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGC DNA Sequence (SEQ ID NO: 5)TCTTTTAAGAGGTGTCCAGTGTCAGGTGCAGCTG GTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAT TCACCTTCAGTAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGC AGTTATATCAAAGGATGGAGGTAATAAATACCATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCA GAGACAATTCCAAGAATGCGCTGTATCTGCAAATGAATAGCCTGAGAGTTGAAGACACGGCTGTGTAT TACTGTGTGAGAAGAGGGCATCAGCTGGTTCTGGGATACTACTACTACAACGGTCTGGACGTCTGGGG CCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCT GCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG GTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTC CTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACA CCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTC GAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGAC CCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGG AGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAAC GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCA AAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAA GAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGA GCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTC CTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGAT GCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA Heavy ChainMEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPG Protein Sequence (SEQ ID NO: 6)RSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVA VISKDGGNKYHADSVKGRFTISRDNSKNALYLQMNSLRVEDTAVYYCVRRGHQLVLGYYYYNGLDVWG QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light Chain DNA ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAASequence (SEQ ID NO: 7) TGCTCTGGGTCTCTGGATCCAGTGGGGATATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCC CTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGTATAGTAATGGATACAACTTTT TGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCT CCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGATTG GAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGG GACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGC AGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGT CTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAG Light ChainMRLPAQLLGLLMLWVSGSSGDIVLTQSPLSLPVTPG Protein Sequence (SEQ ID NO: 8)EPASISCRSSQSLLYSNGYNFLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRLEAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC MatureVariable CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG Domain of Heavy Chain DNAGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG Sequence (SEQ ID NO: 1)CAGCCTCTGGATTCACCTTCAGTAGTTATGGCAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAAGGATGGAGGT AATAAATACCATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATGCGCT GTATCTGCAAATGAATAGCCTGAGAGTTGAAGACACGGCTGTGTATTACTGTGTGAGAAGAGGGCATC AGCTGGTTCTGGGATACTACTACTACAACGGTCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCCTCA Mature VariableQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMH Domain of Heavy Chain ProteinWVRQAPGKGLEWVAVISKDGGNKYHADSVKGRFT Sequence (SEQ ID NO: 2)ISRDNSKNALYLQMNSLRVEDTAVYYCVRRGHQL VLGYYYYNGLDVWGQGTTVTVSS MatureVariable GATATTGTGCTGACTCAGTCTCCACTCTCCCTGCC Domain of Light Chain DNACGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGC Sequence (SEQ ID NO: 3)AGGTCTAGTCAGAGCCTCTTGTATAGTAATGGAT ACAACTTTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTA ATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATC AGCAGATTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTT CGGCCAAGGGACCAAGGTGGAAATCAAA MatureVariable DIVLTQSPLSLPVTPGEPAAISCRSSQSLLYSNGYNFL Domain of Light ChainProtein DWYLQKPGQSPQLLIYLGSNRASGVPDPYSGSGSGT Sequence (SEQ ID NO: 4)DFTLKISRLEAEDVGVYYCMQALQTPRTFGQGTKV EIK Heavy chainCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG DNA (variable domain)GTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG (3.1.1H-A78T) SEQ ID NO: 89CAGCCTCTGGATTCACCTTCAGTAGTTATGGCAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAAGGATGGAGGT AATAAATACCATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATaCGCT GTATCTGCAAATGAATAGCCTGAGAGTTGAAGACACGGCTGTGTATTACTGTGTGAGAAGAGGGCATC AGCTGGTTCTGGGATACTACTACTACAACGGTCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCCTCA Heavy chainQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMH protein (variable domain)WVRQAPGKGLEWVAVISKDGGNKYHADSVKGRFT (3.1.1H-A78T) SEQ ID NO: 90ISRDNSKNTLYLQMNSLRVEDTAVYYCVRRGHQLV LGYYYYNGLDVWGQGTTVTVSS Heavy chainCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG DNA (variable domain)GTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG (3.1.1H-A78T- V88A-V97A)CAGCCTCTGGATTCACCTTCAGTAGTTATGGCAT SEQ ID NO: 91GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTG GAGTGGGTGGCAGTTATATCAAAGGATGGAGGTAATAAATACCATGCAGACTCCGTGAAGGGCCGAT TCACCATCTCCAGAGACAATTCCAAGAATaCGCTGTATCTGCAAATGAATAGCCTGAGAGcTGAAGAC ACGGCTGTGTATTACTGTGcGAGAAGAGGGCATCAGCTGGTTCTGGGATACTACTACTACAACGGTCT GGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA Heavy chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMH protein (variabledomain) WVRQAPGKGLEWVAVISKDGGNKYHADSVKGRFT (3.1.1H-A78T- V88A-V97A)ISRDNSKNTLYLQMNSLRAEDTAVYYCARRGHQLV SEQ ID NO: 92 LGYYYYNGLDVWGQGTTVTVSSLight chain DNA GATATTGTGaTGACTCAGTCTCCACTCTCCCTGCC (variable domain)(3.1.1L-L4M- CGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGC L83V) SEQ ID NO: 93AGGTCTAGTCAGAGCCTCTTGTATAGTAATGGAT ACAACTTTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTA ATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATC AGCAGAgTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTT CGGCCAAGGGACCAAGGTGGAAATCAAA Lightchain DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNF protein (variable domain)LDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSG (3.1.1L-L4M- L83V)TDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTK SEQ ID NO: 94 VEIK

TABLE 4 DNA and protein sequences of antibody 7.1.2 DESCRIPTION:SEQUENCE (signal sequence underlined): Heavy ChainATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGC DNA Sequence (SEQ IDTCTTTTAAGAGGTGTCCAGTGTCAGGTGCAGCTG NO: 13)GTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGG AGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCG CCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAATGATGGAGATAATAAATACCAT GCAGACTCCGTGTGGGGCCGATTCACCATCTCCAGAGACAATTCCAGGAGCACGCTTTATCTGCAAAT GAACAGCCTGAGAGCTGAGGACACGGCTGTATATTACTGTGCGAGAAGAGGCATGGGGTCTAGTGGG AGCCGTGGGGATTACTACTACTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC CTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCA CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC GCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG CGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCA GCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACC ACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC CCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACG TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCC GTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGT CTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAA CCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT GGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC TACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAC AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA CACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA Heavy Chain MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPG Protein Sequence (SEQID RSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVA NO: 14)VISNDGDNKYHADSVWGRFTISRDNSRSTLYLQMN SLRAEDTAVYYCARRGMGSSGSRGDYYYYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light Chain DNA ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAASequence (SEQ ID TGCTCTGGGTCTCTGGATCCAGTGGGGATATTGT NO: 15)GATGACTCAGTCTCCACTCTCCCTGCCCGTCACCC CTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGTATAGTAATGGATACAACTTTT TGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCT CCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTG GAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGG GACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGC AGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGT CTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAG Light ChainMRLPAQLLGLLMLWVSGSSGDIVMTQSPLSLPVTP Protein Sequence (SEQ IDGEPASISCRSSQSLLYSNGYNFLDWYLQKPGQSPQL NO: 16)LIYLGSNRASGVPDRFSGSGSGTDFTKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC MatureVariable CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG Domain of Heavy Chain DNAGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG Sequence (SEQ IDCAGCCTCTGGATTCACCTTCAGTAGCTATGGCAT NO: 9)GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTG GAGTGGGTGGCAGTTATATCAAATGATGGAGATAATAAATACCATGCAGACTCCGTGTGGGGCCGATT CACCATCTCCAGAGACAATTCCAGGAGCACGCTTTATCTGCAAATGAACAGCCTGAGAGCTGAGGACA CGGCTGTATATTACTGTGCGAGAAGAGGCATGGGGTCTAGTGGGAGCCGTGGGGATTACTACTACTAC TACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA Mature Variable QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHDomain of Heavy Chain Protein WVRQAPGKGLEWVAVISNDGDNKYHADSVWGRF Sequence(SEQ ID TISRDNSRSTLYLQMNSLRAEDTAVYYCARRGMGS NO: 10)SGSRGDYYYYYGLDVWGQGTTVTVSS Mature VariableGATATTGTGATGACTCAGTCTCCACTCTCCCTGCC Domain of Light Chain DNACGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGC Sequence (SEQ IDAGGTCTAGTCAGAGCCTCTTGTATAGTAATGGAT NO: 11)ACAACTTTTTGGATTGGTACCTGCAGAAGCCAGG GCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGG CAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATT ACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA Mature VariableDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNF Domain of Light Chain ProteinLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSG Sequence (SEQ IDTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTK NO: 12) VEIK

TABLE 5 DNA and protein sequences of antibody 10.8.3 DESCRIPTION:SEQUENCE (signal sequence underlined): Heavy ChainATGAAACACCTGTGGTTCTTCCTCCTGCTGGTGGC DNA Sequence (SEQ IDAGCTCCCAGATGGGTCCTGTCCCAGGTGCAGCTG NO: 21)CAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGG AGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGATCTGGATCCGGC AGCCCGCCGGGAAGGGACTGGAATGGATTGGGCGTGTCTATACCAGTGGGAGCACCAACTACAACCC CTCCCTCAAGAGTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCT CTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGATGGTCTTTACAGGGGGTACGGTATG GACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCC CCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACT TCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCAC CCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGC AAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCC CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAG CCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACA AAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGG ACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAA AACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAG GAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGT GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT CATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAATGA Heavy ChainMKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSE Protein Sequence (SEQ IDTLSLTCTVSGGSISSYYWIWIRQPAGKGLEWIGRVY NO: 22)TSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAA DTAVYYCARDGLYRGYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light Chain DNA ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGC Sequence (SEQID TGCTCTGGTTCCCAGGTTCCAGATGCGACATCCA NO: 23)GATGACCCAGTCTCCATCTTCCGTGTCTGCATCTG TAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGCCTATTAGCAGCTGGTTAGCCTGGTATCAG CAGAAACCAGGGAAAGCCCCTAAACTCCTGATTTATTCTGCCTCCGGTTTGCAAAGTGGGGTCCCATC AAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATT TTGCAACTTACTATTGTCAACAGACTGACAGTTTCCCGCTCACTTTCGGCGGCGGGACCAAGGTGGAGA TCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGA TAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC AGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG Light ChainMRLPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVG Protein Sequence (SEQ IDDRVTITCRASQPISSWLAWYQQKPGKAPKLLIYSAS NO: 24)GLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTDSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC Mature VariableCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTG Domain of Heavy Chain DNAGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCA Sequence (SEQ IDCTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGG NO: 17)ATCTGGATCCGGCAGCCCGCCGGGAAGGGACTG GAATGGATTGGGCGTGTCTATACCAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCAC CATGTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGG CCGTGTATTACTGTGCGAGAGATGGTCTTTACAGGGGGTACGGTATGGACGTCTGGGGCCAAGGGAC CACGGTCACCGTCTCCTCA Mature VariableQVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWIWI Domain of Heavy Chain ProteinRQPAGKGLEWIGRVYTSGSTNYNPSLKSRVTMSVD Sequence (SEQ IDTSKNQFSLKLSSVTAADTAVYYCARDGLYRGYGM NO: 18) DVWGQGTTVTVSS Mature VariableGACATCCAGATGACCCAGTCTCCATCTTCCGTGT Domain of Light Chain DNACTGCATCTGTAGGAGACAGAGTCACCATCACTTG Sequence (SEQ IDTCGGGCGAGTCAGCCTATTAGCAGCTGGTTAGCC NO: 19)TGGTATCAGCAGAAACCAGGGAAAGCCCCTAAA CTCCTGATTTATTCTGCCTCCGGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGG ACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGAC TGACAGTTTCCCGCTCACTTTCGGCGGCGGGACCAAGGTGGAGATCAAA Mature Variable DIQMTQSPSSVSASVGDRVTITCRASQPISSWLAWYDomain of Light Chain Protein QQKPGKAPKLLIYSASGLQSGVPSRFSGSGSGTDFTSequence (SEQ ID LTISSLQPEDFATYYCQQTDSFPLTFGGGTKVEIK NO: 20)

TABLE 6 DNA and protein sequences of antibody 15.1.1 DESCRIPTION:SEQUENCE (signal sequence underlined): Heavy ChainATGAAACATCTGTGGTTCTTCCTTCTCCTGGTGGC DNA Sequence (SEQ IDAGCTCCCAGATGGGTCCTGTCCCAGGTGCAGCTG NO: 29)CAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGG AGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAAGTTACTACTGGACCTGGATCCGGC AGCCCCCAGGGAAGGGACTGGAGTGGATTGGATATATCTATTACAGTGGGAGCACCAACTACAATCC CTCCCTCAAGAGTCGAGTCACCATATCAGTAGACATGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTT CTGTGACCGCTGCGGACACGGCCGTTTATTACTGTGCGAGAAAGGGTGACTACGGTGGTAATTTTAAC TACTTTCACCAGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGT CTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTT CCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTT CGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTT GAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCT CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGG ACGTGAGTCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGC ACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCAT CGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC CGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGA CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAATGA Heavy ChainMKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSE Protein Sequence (SEQ IDTLSLTCTVSGGSIRSYYWTWIRQPPGKGLEWIGYIY NO: 30)YSGSTNYNPSLKSRVTISVDMSKNQFSLKLSSVTAA DTAVYYCARKGDYGGNFNYFHQWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light Chain DNA ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAA Sequence (SEQID TGCTCTGGGTCTCTGGATCCAGTGGGGATATTGT NO: 31)GATGACTCAGTCTCCACTCTCCCTGCCCGTCACCC CTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTACATACTAATGGATACAACTAT TTCGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAACTCCTGATCTATTTGGGTTCTAATCGGGCC TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGT GGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACAGTTTTGGCCAGG GGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGT CTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAG Light ChainMRLPAQLLGLLMLWVSGSSGDIVMTQSPLSLPVTP Protein Sequence (SEQ IDGEPASISCRSSQSLLHTNGYNYFDWYLQKPGQSPQL NO: 32)LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYSFGQGTKLEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC MatureVariable CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTG Domain of Heavy Chain DNAGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCA Sequence (SEQ IDCTGTCTCTGGTGGCTCCATCAGAAGTTACTACTG NO: 25)GACCTGGATCCGGCAGCCCCCAGGGAAGGGACT GGAGTGGATTGGATATATCTATTACAGTGGGAGCACCAACTACAATCCCTCCCTCAAGAGTCGAGTCA CCATATCAGTAGACATGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCTGCGGACACG GCCGTTTATTACTGTGCGAGAAAGGGTGACTACGGTGGTAATTTTAACTACTTTCACCAGTGGGGCCA GGGAACCCTGGTCACCGTCTCCTCA MatureVariable QVQLQESGPGLVKPSETLSLTCTVSGGSIRSYYWTW Domain of Heavy ChainProtein IRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVD Sequence (SEQ IDMSKNQFSLKLSSVTAADTAVYYCARKGDYGGNFN NO: 26) YFHQWGQGTLVTVSS MatureVariable GATATTGTGATGACTCAGTCTCCACTCTCCCTGCC Domain of Light Chain DNACGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGC Sequence (SEQ IDAGGTCTAGTCAGAGCCTCCTACATACTAATGGAT NO: 27)ACAACTATTTCGATTGGTACCTGCAGAAGCCAGG GCAGTCTCCACAACTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGG CAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATT ACTGCATGCAAGCTCTACAAACTCCGTACAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAA Mature VariableDIVMTQSPLSLPVTPGEPASISCRSSQSLLHTNGYNY Domain of Light Chain ProteinFDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSG Sequence (SEQ IDTDFTLKISRVEAEDVGVYYCMQALQTPYSFGQGTK NO: 28) LEIK

TABLE 7 DNA and protein sequences of antibody 21.4.1 DESCRIPTION:SEQUENCE (signal sequence underlined): Heavy ChainATGGACTGGACCTGGAGGATCCTCTTCTTGGTGG DNA Sequence (SEQ IDCAGCAGCCACAGGAGCCCACTCCCAGGTGCAGCT NO: 45)GGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGG GGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGC GACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAACTA TGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGC TGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATCAGCCCCTAGGATATTGT ACTAATGGTGTATGCTCCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTC CACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCC TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACC AGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGAC CGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCA AGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCA GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT CACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGC GTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCA GCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA AGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTG TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGG TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA GAGCCTCTCCCTGTCTCCGGGTAAATGA HeavyChain MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPG Protein Sequence (SEQ IDASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM NO: 46)GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMEL NRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light Chain DNA ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGCSequence (SEQ ID TGCTCTGGTTCCCAGGTTCCAGATGCGACATCCA NO: 47)GATGACCCAGTCTCCATCTTCCGTGTCTGCATCTG TAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTTACAGCTGGTTAGCCTGGTATCAG CAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATACTGCATCCACTTTACAAAGTGGGGTCCCATC AAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATT TTGCAACTTACTATTGTCAACAGGCTAACATTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGA TCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGA TAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC AGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG Light ChainMRLPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVG Protein Sequence (SEQ IDDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA NO: 48)STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC Mature VariableCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA Domain of Heavy Chain DNAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAA Sequence (SEQ IDGGCTTCTGGATACACCTTCACCGGCTACTATATG NO: 41)CACTGGGTGCGACAGGCCCCTGGACAAGGGCTTG AGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTC ACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACA CGGCCGTGTATTACTGTGCGAGAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTG ACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA Mature Variable QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYM Domain of HeavyChain Protein HWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGR Sequence (SEQ IDVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPL NO: 42) GYCTNGVCSYFDYWGQGTLVTVSSMature Variable GACATCCAGATGACCCAGTCTCCATCTTCCGTGT Domain of Light ChainDNA CTGCATCTGTAGGAGACAGAGTCACCATCACTTG Sequence (SEQ IDTCGGGCGAGTCAGGGTATTTACAGCTGGTTAGCC NO: 43)TGGTATCAGCAGAAACCAGGGAAAGCCCCTAAC CTCCTGATCTATACTGCATCCACTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGG ACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCAACTTACTATTGTCAACAGGC TAACATTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA Mature Variable DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYDomain of Light Chain Protein QQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTSequence (SEQ ID LTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIK NO: 44)

TABLE 8 DNA and protein sequences of mature variable domains of 21.2.1antibody DESCRIPTION: SEQUENCE: Heavy ChainCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG DNA (SEQ IDGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG NO: 33)CAGCCTCTGGATTCACCTTCAGTAGCTATGTCAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAGTA GTAAATACTATGCAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATAAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGGGGTAA AGCAGTGCCTGGTCCTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAG Heavy Chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYVMProtein (SEQ ID HWVRQAPGKGLEWVAVMSYDGSSKYYANSVKGRF NO: 34)TISRDNSKNTLYLQINSLRAEDTAVYYCARDGGK AVPGPDYWGQGILVTVSS Light Chain DNAGATATTGTGATGACTCAGTCTCCACTCTCCCTGC (SEQ ID NO: 35)CCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTG CAGGTCTAGTCAGAGTGTTCTGTATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAG GGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGT GGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTA TTACTGCATGCAAGTTTTACAAACTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC Light ChainDIVMTQSPLSLPVTPGEPASISCRSSQSVLYSNG Protein (SEQ IDYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFS NO: 36)GSGSGTDFTLKISRVEAEDVGVYYCMQVLQTPFT FGPGTKVDIK

TABLE 9 DNA and protein sequences of mature variable domains of 22.1.1antibody DESCRIPTION: SEQUENCE: Heavy ChainCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG DNA (SEQ IDGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG NO: 49)CAGCCTCTGGATTCACCTTCAGTCGCTATGGCAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATCTGATGGAGGTA ATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTACGAGAAGAGGGACTGG AAAGACTTACTACCACTACTGTGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG Heavy ChainQVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGM Protein (SEQ IDHWVRQAPGKGLEWVAVISSDGGNKYYADSVKGRF NO: 50)TISRDNSKNTLYLQMNSLRAEDTAVYYCTRRGTG KTYYHYCGMDVWGQGTTVTVSS Light ChainDNA GATATTGTGATGACTCAGTCTCCACTCTCCCTGC (SEQ ID NO: 51)CCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTG CAGGTCTAGTCAGAGCCTCCTGTATAGTAATGGATATAACTATTTGGATTGGTACCTGCAGAAGCCAG GGCAGTCTCCACACCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGT GGCAGTGGTTCAGGCACTGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTA TTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC Light ChainDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNG Protein (SEQ IDYNYLDWYLQKPGQSPHLLIYLGSNRASGVPDRFS NO: 52)GSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRT FGQGTKVEIK

TABLE 10 DNA and protein sequences of mature variable domains of 23.5.1antibody DESCRIPTION: SEQUENCE: Heavy ChainCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG DNA (SEQ IDGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG NO: 57 )TAGCCTCTGGATTCACCTTCAGTAACTATGGCAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAATTATATCATATGATGGAAGTA ATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATGTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGACGCGGTCACT ACGGGAGGGATTACTACTCCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC TCAG Heavy ChainQVQLVESGGGVVQPGRSLRLSCVASGFTFSNYGM Protein (SEQ IDHWVRQAPGKGLEWVAIISYDGSNKYYADSVKGRF NO: 58)TISRDNSKNTLYVQMNSLRAEDTAVYYCARRGHY GRDYYSYYGLDVWGQGTTVTVSS Light ChainDNA GATATTGTGATGACTCAGTCTCCACTCTCCCTGC (SEQ ID NO: 59)CCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTG CAGGTCTAGTCAGAGCCTCCTGCCTGGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAG GGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGT GGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTA TTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC Light ChainDIVMTQSPLSLPVTPGEPASISCRSSQSLLPGNG Protein (SEQ IDYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFS NO: 60)GSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRT FGQGTKVEIK

TABLE 11 DNA and protein sequences of mature variable domains of 23.28.1antibody DESCRIPTION: SEQUENCE: Heavy ChainCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTG DNA (SEQ IDGTGAAGCCTTCGGACACCCTGTCCCTCACCTGCA NO: 65)CTGTCTCTGGTGGCTCCATCAGAGGTTACTACTG GAGCTGGATCCGGCAGCCCCCTGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGC ACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTC CCTGAAGCTGAACTCTGTGACCGCTGCGGACACGGCCGTGTATTATTGTGCGAGAAAGGGGGGCCTCT ACGGTGACTACGGCTGGTTCGCCCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG Heavy ChainQVQLQESGPGLVKPSDTLSLTCTVSGGSIRGYYWS Protein (SEQ IDWIIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISV NO: 66)DTSKNQFSLKLNSVTAADTAVYYCARKGGLYGDY GWFAPWGQGTLVTVSS Light ChainGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGT DNA (SEQ IDCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTG NO: 67)CAGGGCCAGTCAGAGTGTTAGCAGCAGCGACTTA GCCTGGCACCAGCAGAAACCTGGCCAGGCTCCCAGACTCCTCATCTATGGTGCATCCAGCAGGGCCAC TGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGG AGCCTGAAGATTTTGCAGTGTATTACTGTCAGCACTGTCGTAGCTTATTCACTTTCGGCCCTGGGACCA AAGTGGATATCAAAC Light ChainEIVLTQSPGTLSLSPGERATLSCRASQSVSSSDLAWH Protein (SEQ IDQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTL NO: 68)TISRLEPEDFAVYYCQHCRSLFTFGPGTKVDIK Heavy ChainCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTG DNA (variable domain)GTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCA (23.28.1H- D16E) (SEQCTGTCTCTGGTGGCTCCATCAGAGGTTACTACTG ID NO: 97)GAGCTGGATCCGGCAGCCCCCTGGGAAGGGACT GGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCA CCATATCAGTAGACACGTCCAAGAACOAGTTCTCCCTGAAGCTGAACTCTGTGACCGCTGCGGACACG GCCGTGTATTATTGTGCGAGAAAGGGGGGCCTCTACGGTGACTACGGCTGGTTCGCCCCCTGGGGCCA GGGAACCCTGGTCACCGTCTCCTCAG HeavyChain QVQLQESGPGLVKPSLTLSLTCTVSGGSIRGYYWS Protein (var- iable domain)WIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISV (23.28.1H- D16E) (SEQDTSKNQFSLKLNSVTAADTAVYYCARKGGLYGDY ID NO: 98) GWFAPWGQGTLVTVSS.

TABLE 12 DNA and protein sequences of mature variable domains of 23.29.1antibody DESCRIPTION: SEQUENCE: Heavy ChainCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTG DNA (SEQ IDGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTG NO: 73)CAGCCTCTGGATTCACCTTCAGTAGCTATGCCAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTA ATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTACAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGACGCGGTCACTA CGGGAATAATTACTACTCCTATTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCT CAG Heavy ChainQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMH Protein (SEQ IDWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFT NO: 74)IYRDNSKNTLYLQMNSLRAEDTAVYYCARRGHYG NNYYSYYGLDVWGQGTTVTVSS Light ChainGATATTGTGATGACTCAGTCTCCACTCTCCCTGCC DNA (SEQ IDCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGC NO: 75)AGGTCTAGTCAGAGCCTCCTGCCTGGTAATGGAT ACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTA ATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGCTCAGGCACAGATTTTACACTGAAAATC AGCAGAGTGGAGGCTGAGGATGTTGGGATTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTT CGGCCAAGGGACCAAGGTGGAAATCAAAC LightChain DIVMTQSPLSLPVTPGEPASISCRSSQSLLPGNGYNY Protein (SEQ IDLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSG NO: 76)TDFTLKISRVEAEDVGIYYCMQALQTPRTFGQGTK VEIK

TABLE 13 DNA and protein sequences of mature variable domains of 24.2.1antibody DESCRIPTION: SEQUENCE Heavy ChainCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTG DNA (SEQ IDGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCA NO: 81)CTGTCTCTGGTGGCTCCATCAGAGGTTACTACTG GAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGC ACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTC CCTGAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAAGGGGGGGCCTCT ACGGTGACTACGGCTGGTTCGCCCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG Heavy ChainQVQLQESGPGLVKPSETLSLTCTVSGGSIRGYYWS Protein (SEQ IDWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISV NO: 82)DTSKNQFSLKLSSVTAADTAVYYCARRGGLYGDY GWFAPWGQGTLVTVSS Light ChainGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGT DNA (SEQ IDCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTG NO: 83)CAGGGCCAGTCAGAGTGTTAGCAGCACCTACTTA GCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCAC TGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGG AGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATAGTAGCTTATTCACTTTCGGCCCTGGGACC AAAGTGGATATCAAAC Light ChainETVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWY Protein (SEQ IDQQKPGQAPRLLIYGASSRATGIIPDRFSGSGSGTDFT NO: 84)LTISRLEPEDFAVYYCQQYSSLFTFGPGTKVDIK

TABLE 14 DNA and protein sequences of antibody 21.2.1 SEQUENCE (signalDESCRIPTION: sequence underlined): Heavy ChainATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGC DNA (SEQ IDTCTTTTAAGAGGTGTCCAGTGTCAGGTGCAGCTG NO: 37)GTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGG AGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGTCATGCACTGGGTCCG CCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAGTAGTAAATACTAT GCAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAAT AAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGGGGTAAAGCAGTGCCTG GTCCTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTC TTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGG ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTC CCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTC GGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTG AGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGA CGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA AGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCAC CAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCG AGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG GGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACT CCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAATGA Heavy ChainMEFGLSWVFLVALLRGVOCQVQLVESGGGVVQPG Protein (SEQ IDRSLRLSCAASGFTFSSYVMHWVRQAPGKGLEWVA NO: 38)VMSYDGSSKYYANSVKGRIFTISRDNSKNTLYLQINS LRAEDTAVYYCARDGGKAVPGPDYWGQGILVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSNTGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light Chain ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAA DNA (SEQ IDTGCTCTGGGTCTCTGGATCCAGTGGGGATATTGT NO: 39)GATGACTCAGTCTCCACTCTCCCTGCCCGTCACCC CTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGTGTTCTGTATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCC TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGT GGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGTTTTACAAACTCCATTCACTTTCGGCCCTGG GACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCA GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACA GTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAG GACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCT ACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG TTAG Light ChainMRLPAQLLGLLMLWVSGSSGDIVMTQSPLSLPVTP Protein (SEQ IDGEPASISCRSSQSVLYSNGYNYLDWYLQKPGQSPQL NO: 40)LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQVLQTPFTFGPGTKVDWRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC

TABLE 15 DNA and protein sequences of antibody 22.1.1 SEQUENCE (signalDESCRIPTION: sequence underlined): Heavy ChainATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGC DNA (SEQ IDTCTTTTAAGAGGTGTCCAGTGCAGGTGCAACTG NO: 53)GTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGG AGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTCGCTATGGCATGCACTGGGTCCG CCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATCTGATGGAGGTAATAAATACTAT GCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAAT GAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTACGAGAAGAGGGACTGGAAAGACTTACT ACCACTACTGTGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAG GGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGC GTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCC CTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTG GACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACC GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGT GCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTC CTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCC TCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC CCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCT ACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACAC CTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGA GCCTCTCCCTGTCTCCGGGTAAATGA Heavy ChainMEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPG Protein (SEQ IDRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVA NO: 54)VISSDGGNKYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCTRRGTGKTYYHYCGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVTHNAKTKPRE EQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPTEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light Chain ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAA DNA (SEQ IDTGCTCTGGGTCTCTGGATCCAGTGGGGATATTGT NO: 55)GATGACTCAGTCTCCACTCTCCCTGCCCGTCACCC CTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGTATAGTAATGGATATAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACACCTCCTGATCTATTTGGGTTCTAATCGGGCC TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGTTCAGGCACTGATTTTACACTGAAAATCAGCAGAGT GGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAG GGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCYTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGT CTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAG Light ChainMRLPAOLLGLLMLWVSGSSGDIVMTQSPLSLPVTP Protein (SEQ IDGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPHL NO: 56)LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFWP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC

TABLE 16 DNA and protein sequences of antibody 23.5.1 DESCRIPTION:SEQUENCE (signal sequence underlined): Heavy ChainATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGC DNA (SEQ IDTCTTTTAAGAGGTGTCCAGTGTCAGGTGCAGCTG NO: 61)GTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGG AGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCACCTTCAGTAACTATGGCATGCACTGGGTCCGC CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAATTATATCATATGATGGAAGTAATAAATACTATG CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATGTGCAAATG AACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGACGCGGTCACTACGGGAGGGATTA CTACTCCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCA AGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGG CTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCG GCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG CCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGG TGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCAC GTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGG AGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGT CCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACA CCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC TACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACA CCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGA GCCTCTCCCTGTCTCCGGGTAAATGA HeavyChain MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPG Protein (SEQ IDRSLRLSCVASGFTFSNYGMHWVRQAPGKGLEWVA NO: 62)IISYDGSNKYYADSVKGRETISRDNSKNTLYVQMNS LRAEDTAVYYCARRGHYGRDYYSYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG LPAPIEKTLSKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light Chain DNA ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAA (SEQ IDNO: 63) TGCTCTGGGTCTCTGGATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCC CTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCCTGGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCC TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGT GGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAG GGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG CAGTTGAAATCTGGAACTGCCTSTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCYTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGT CTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAA Light ChainMRLPAQLLGLLMLWVSGSSGDIVMTQSPLSLPVTP Protein (SEQ IDGEPASISCRSSQSLLPGNGYNYLDWYLQKPGQSPQL NO: 64)LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTAXVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC

TABLE 17 DNA and protein sequences of antibody 23.28.1 DESCRIPTION:SEQUENCE (signal sequence underlined): Heavy ChainATGAAACATCTGTGGTTCTTCCTTCTCCTGGTGGC DNA (SEQ IDAGCTCCCAGATGGGTCCTGTCCCAGGTGCAGCTG NO: 69)CAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGG AGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAGGTTACTACTGGAGCTGGATCCGGC AGCCCCCTGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCC CTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAACT CTGTGACCGCTGCGGACACGGCCGTGTATTATTGTGCGAGAAAGGGGGGCCTCTACGGTGACTACGG CTGGTTCGCCCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGG TCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTT CCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTT CGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTT GAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCT CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGG ACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGC ACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCAT CGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC CGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGA CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAATGA Heavy ChainMKIHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSE Protein (SEQ IDTLSLTCTVSGGSIRGYYWSWRQPPGKGLEWIGYIY NO: 70)YSGSTNYNPSLKSRVTISVDTSKNQFSLKLNSVTAA DTAVYYCARKGGLYGDYGWFAPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light Chain DNA ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCT (SEQ ID NO:71) ACTCTGGCTCCCAGAATCCACCGGAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCC AGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCGACTTAGCCTGGCACC AGCAGAAACCTGGCCAGGCTCCCAGACTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGA TTTTGCAGTGTATTACTGTCAGCACTGTCGTAGCTTATTCACTTTCGGCCCTGGGACCAAAGTGGATAT CAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGAT AACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACA GCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGT CACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG Light ChainMETPAQLLFLLLLWLPESTGEIVLTQSPGTLSLSPGE Protein (SEQ IDRATLSCRASQSVSSSDLAWHQQKPGQAPRLLIYGA NO: 72)SSRATGTPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHCRSLFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

TABLE 18 DNA and protein sequences of antibody 23.29.1 DESCRIPTION:SEQUENCE (signal sequence underlined): Heavy ChainATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGC DNA (SEQ IDTCTTTTAAGAGGTGTCCAGTGTCAGGTGCAACTG NO: 77)GTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGG AGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCCATGCACTGGGTCCG CCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTAT GCAGACTCCGTGAAGGGCCGATTCACCATCTACAGAGACAATTCCAAGAACACGCTGTATCTGCAAAT GAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGACGCGGTCACTACGGGAATAATT ACTACTCCTATTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACC AAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGG GCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGC GGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGT GCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAG GTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGG ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCA CGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCG TCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGG CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT CTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACA CCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGA GCCTCTCCCTGTCTCCGGGTAAATGA HeavyChain MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPG Protein (SEQ IDRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVA NO: 78)VISYDGSNKYYADSVKGRFTIYRDNSKNTLYLQMN SLRAEDTAVYYCARRGHYGNNYYSYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSNEGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNK GLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPM LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light Chain DNA ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAA (SEQID NO: 79) TGCTCTGGGTCTCTGGATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCC CTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCCTGGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCC TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGCTCAGGCACAGATTTTACACTGAAAATCAGCAGAGT GGAGGCTGAGGATGTTGGGATTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAG GGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTT CAGTGGAGGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGT CTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAG Light ChainMRLPAQLLGLLMLWVSGSSGDIVMTQSPLSLPVTP Protein (SEQ IDGEPASISCRSSQSLLPGNGYNYLDWYLQKPGQSPQL NO: 80)LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWRVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC LightChain DNA ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAA (23.29.1LR174K) (SEQ IDNO:101) TGCTCTGGGTCTCTGGATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCC CTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCCTGGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCC TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGCTCAGGCACAGATTTTACACTGAAAATCAGCAGAGT GGAGGCTGAGGATGTTGGGATTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAG GGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTT CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGT CTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAG Light ChainMRLPAQLLGLLMLWVSGSSGDIVMTQSPLSLPVTP Protein (23.29.1LR174K)GEPASISCRSSQSLLPGNGYNYLDWYLQKPGQSPQL (SEQ ID NO:1O1)LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIIFP PSDEQLKSGTASVVCLLNMYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC

TABLE 19 DNA and protein sequences of antibody 24.2.1 DESCRIPTION:SEQUENCE (signal sequence underlined): Heavy ChainATGAAACATCTGTGGTTCTTCCTTCTCCTGGTGGC DNA (SEQ IDAGCTCCCAGATGGGTCCTGTCCCAGGTGCAGCTG NO: 85)CAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGG AGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAGGTTACTACTGGAGCTGGATCCGGC AGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCC CTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTT CTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAAGGGGGGGCCTCTACGGTGACTACGG CTGGTTCGCCCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGG TCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTT CCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTT CGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTT GAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCT CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGG ACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGC ACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCAT CGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCC CGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGA CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAATGA Heavy ChainMKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSE Protein (SEQ IDTLSLTCTVSGGSIRGYYWSWIRQPPGKGLEWIGYW NO: 86)YSGSTNYNIPSLKSRVTJSVDTSKNQFSLKISSVTAA DTAVYYCARRGGLYGDYGWFAPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDJAVEWESNGQPENNYKTTPPMLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNIIYTQKSLSLSPGK Light Chain DNA ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCT (SEQ ID NO:87) ACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCC AGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCACCTACTTAGCCTGGTACC AGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGA TTTTGCAGTGTATTACTGTCAGCAGTATAGTAGCTTATTCACTTTCGGCCCTGGGACCAAAGTGGATAT CAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGAT AACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACA GCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGT CACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG Light ChainMETPAOLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGE Protein (SEQ IDRATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGA NO: 88)SSRATGTPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSLFTFGPGTKVD1KRTVAAPSVFWPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

TABLE 20 DNA and protein sequences of the mature variable domains ofantibody 22.1.1 H-C109A SEQUENCE (signal sequence DESCRIPTION:underlined): Heavy Chain CAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTG DNA (SEQ IDNO:95) GTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTCGCTATGGCAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATCTGATGGAGGTA ATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTACGAGAAGAGGGACTGG AAAGACTTACTACCACTACGCCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG Heavy ChainQVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGM Protein (SEQ ID NO:96)HWVRQAPGKGLEWVAVISSDGGNKYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCTRRGTGKTYYHYAGMDVWGQGTTVTVSS

TABLE 21 DNA and protein sequences of the mature variable domains ofantibody 23.28.1 L-C92A SEQUENCE (signal sequence DESCRIPTION:underlined): Light Chain DNA GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGT (SEQ IDNO:99) CTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCGACTTA GCCTGGCACCAGCAGAAACCTGGCCAGGCTCCCAGACTCCTCATCTATGGTGCATCCAGCAGGGCCAC TGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGG AGCCTGAAGATTTTGCAGTGTATTACTGTCAGCACGCCCGTAGCTTATTCACTTTCGGCCCTGGGACC AAAGTGGATATCAAAC Light ChainEIVLTQSPGTLSLSPGERATLSCRASQSVSSSDL Protein (SEQ ID NO:100)AWHQQKPGQAPRLLIYGASSRATGIPDRYSGSGS GTDFTLTISRLEPEDFAVYYCQHARSLFTFGPGTKVDIK

EXAMPLE III Analysis of Heavy and Light Chain Amino Acid Substitutions

FIGS. 1D-1H and 2D-2H provide sequence alignments between the predictedheavy chain variable domain amino acid sequences of monoclonalantibodies 3.1.1, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1,22.1.1H-C109A, 23.5.1, 23.28.1, 23.28.1H-D16E, 23.29.1 and 24.2.1antibodies and the germline amino acid sequences of their respectivegenes. Most of the heavy chain CDR3 regions contain amino acidinsertions.

The DLR1 gene used in the V_(H) domain of antibody 21.4.1 codes for twocysteine (Cys) residues. Mass spectrometry analysis and homologymodeling demonstrated that the two Cys residues are disulfide-linked,and that this disulfide link does not disrupt the structure of theantibody.

FIGS. 1A-1C and 2A-2C provide sequence alignments between the predictedlight chain variable amino acid sequences of monoclonal antibodies3.1.1, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.28.1,23.28.1L-C92A, 23.29.1 and 24.2.1 clones and the germline amino acidsequences of their respective genes. The light chains of theseantibodies are derived from three different Vκ genes. Seven of theeleven antibodies use the A3/A19 Vκ gene, six of which have twomutations in the CDR1 region. Further, five of the seven antibodies thatuse the A3/A19 Vκ gene, also use the Jκ1 gene; in all of theseantibodies the first amino acid derived from the Jκ1 gene isconsistently changed from a W to an R.

It will be appreciated that many of the above-identified amino acidsubstitutions or insertions exist in close proximity to or within a CDR.Such substitutions would appear to bear some effect upon the binding ofthe antibody to the CD40 molecule. Further, such substitutions couldhave significant effect upon the affinity of the antibodies.

EXAMPLE IV Species Crossreactivity of the Antibodies of the Invention

We performed FACS analyses to determine the binding and affinity of theantibodies of the invention for CD40 from various species, particularlycertain old world monkeys. We incubated aliquots of human and monkeywhole blood for 1 hour on ice with increasing concentrations ofanti-CD40 antibodies of the invention exemplified herein or with ananti-keyhole limpet hemocyanin (KLH) antibody as a negative control. Wethen incubated the samples for 30 minutes on ice with anti-humanIgG2-conjugated RPE (phycoerythrin). We measured binding by flowcytometry of CD19/CD20 positive B cells and analyzed the histograms offluorescence intensity (F12-H) versus cell number (Counts) usingCellQuest software. We estimated binding (K_(D)) for each antibody fromgraphs of mean fluorescence intensity versus antibody concentration. Wecontrolled for depletion of the antibody by measuring binding over arange of cell concentrations.

We tested antibodies 3.1.1, 7.1.2, 10.8.3, 15.1.1 and 21.4.1 for bindingto human, rhesus and cynomolgus B cells. We also tested antibodies21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1, 23.29.1 and 24.2.1 for bindingto human and cynomolgus B cells.

We observed that the maximum signal and the concentration for halfmaximum binding to monkey cells, was within a factor of two to thecorresponding parameters for human B cells. No binding was observed insimilar experiments with mouse, rat, rabbit and dog blood.

EXAMPLE V Selectivity of Antibodies for CD40

We conducted another in vitro assay to determine the selectivity ofantibodies of the invention with respect to CD40.

CD40 Selectivity ELISA: Materials and Methods

We coated a 96-well FluoroNUNC plate (Nunc Cat No. 475515) with fourantigens: CD40/Ig, CD44/Ig, RANK/Ig, 4-1BB/Ig, TNFR-1/Ig and TNFR-2/Ig(antigens generated in-house), overnight at +4° C. at 1 μg/ml of 100μl/well in 0.1 M sodium bicarbonate buffer, pH 9.6. We then washed theplate with PBST (PBS+0.1% Tween-20) three times and blocked the platewith PBST+0.5% BSA at 150 μl/well. We incubated the plate at roomtemperature for 1 hour and then washed with PBST three times. Next, wediluted the anti-CD40 antibodies generated in Example I in block at 1μg/ml and added the diluted antibodies to the plate. We incubated theplate at room temperature for 1 hour then washed with PBST three times.We then treated the wells that contained the antibodies generated inExample I with 100 ml/well anti-human IgG2-HRP (Southern Biotech CatNo.9070-05) at a 1:4000 dilution in block. Also, we treated one row withanti-human IgG (Jackson Cat No. 209-035-088) diluted to 1:5000 in blockand added at 1001/well to normalize for plate coating. We also treatedone row with anti-human CD40-HRP (Pharmingen Cat No. 345815/Custom HRPconjugated) at 0.05 μg/ml diluted in block as a positive control. Weincubated the plate at room temperature for 1 hour and then washed withPBST three times. We added TMB substrate (K & P Labs) at 1001/well andincubated the plate for 5 to 10 minutes. We then read the plate using aSpectra-Max™ plate reader. The results showed that the antibodies have aselectivity for CD40 that is at least 100 times greater than theirselectivity for RANK, 4-1BB, TNFR-1 and TNFR-2 in that the CD4 specificsignal (CD40 signal minus background) is at least 100× greater than thecorresponding signal for the other molecules.

EXAMPLE VI Epitope Classification Studies

Having demonstrated that the antibodies of the invention are selectivefor CD40, we performed competition binding analysis using BIAcore andFACS.

BIAcore Competition Studies

We conducted BIAcore competition studies to determine whether the humananti-CD40 antibodies of the invention bind to the same or distinct siteson the CD40 molecule.

In these experiments we used a BIAcore 2000 instrument, following themanufacturer's protocols. Protein-A was immobilized on the sensor chipsurfaces of the BIAcore. A saturating concentration of CD40-Ig whichcomprises the extracellular domain of CD40 was bound to the sensorchip.We then bound a first human agonist anti-CD40 antibody of the invention,a commercial anti-CD40 antibody or CD40L to the sensorchip-bound CD40under saturating conditions. We then measured the ability of a secondhuman agonist anti-CD40 antibody of the invention to compete with thefirst antibody, commercial antibody or CD40L for binding to CD40. Thistechnique enabled us to assign the antibodies to different bindinggroups. Binding to CD40 indicated recognition of an independent epitope.Lack of binding may indicate recognition of the same epitope oroverlapping epitopes.

FACS Studies

We conducted FACS studies to determine whether the human anti-CD40antibodies of the invention bind to the same or distinct sites on theCD40 molecule, and to determine whether they bind to the same ordistinct site on the CD40 molecule as commercially available anti-CD40antibodies EA5 (Alexis Cat. No. ANC-300-050), LOB7/6 (Serotec MCA/590PE)and 5C3 (Pharmingen # 555458 (unlabeled) and 555460 (PE labeled forFACS).

We counter-stained dendritic cells treated with anti-CD40 antibodies ofthe invention with PE labeled EA5 or PE labeled LOB7/6 antibody on icefor 30 minutes. After a wash, cell staining was analyzed on a B-Dcaliber cytometer. Reduced binding of the commercial antibodies wasinterpreted as an indication that the test antibody bound to the same oroverlapping epitope.

Competition binding analysis by BIAcore and FACS showed that theepitopes recognized by mAb 21.4.1 antibodies overlaps with the epitoperecognized by the EA5 antibody, did not overlap with the epitoperecognized by the commercially available LOB7/6 antibody and does notoverlap with the binding site for CD40L. The epitopes recognized by theremaining antibodies do overlap with the binding site for CD40L.

Table 22 summarizes the results of these epitope classification studies.

TABLE 22 BIAcore Competition Epitope Classification of Certain Anti-CD40Antibodies Of The Invention 3.1.1, 21.2.1, 22.1.1, 23.25.1, 23.5.1,23.28.1, EA5 5C3 LOB7/6 23.29.1 21.4.1 24.2.1 CD40L EA5 X X X X 5C3 X XX X X LOB7/6 X X X X 3.1.1, X X X 21.2.1, 22.1.1, 23.5.1, 23.29.1 21.4.1X X X 23.25.1, X X X X 23.28.1, 24.2.1 CD40L X X X X X X

EXAMPLE VII Upregulation of Surface Molecules by Anti-CD40 Antibodies

We conducted a whole blood assay to determine whether the humananti-CD40 antibodies of the invention upregulate the expression ofsurface molecules on B cells.

Human or primate whole blood was diluted 1:1 with RPMI medium andincubated 24 hours with various concentrations of CD40 agonistantibodies or controls. Cells were stained for 30 minutes (on ice, inthe dark) for HLA-DR, ICAM, B7-1, B7-2, CD19/CD20, CD40, CD23 and CD71,using commercially available, fluorochrome labeled antibody reagents.The cells were then analyzed on a FACSCalibur™ (Becton-Dickinson).B-cells were identified by gating on CD19 or CD20 positive cells, andactivation markers determined for this gate.

The maximum fold increase of median fluorescence (at ≦1 μg/ml antibody),and mean EC₅₀ obtained using one of the anti-CD40 antibodies of theclaimed invention (21.4.1) are shown in Table 23.

TABLE 23 Upregulation of B-Cell Surface Molecules by an Anti-CD40Antibody of the Invention Maximum Fold Increase EC₅₀ (ng/ml) Mean +/−St. Dev. Mean +/− St. Dev. MHC II 4.50 +/− 0.52 3.85 +/− 0.35 CD71 2.30+/− 0.77 0.73 +/− 0.28 ICAM 4.52 +/− 2.42 15.3 +/− 7.3 CD23 69.9 +/−25.8 19.0 +/− 4.4 B7-2 2.74 +/− 0.14 16.0 +/− 21.9

We also conducted experiments to determine whether the human anti-CD40antibodies of the invention upregulate the expression of surfacemolecules of monocyte-derived dendritic cell.

Preparation of the Monocyte Derived Dendritic Cells

Peripheral blood was collected from normal human volunteers. Mononuclearcells were isolated using Sigma ACCUSPIN™ tubes (St. Louis, Mo.), washedwith RPMI media (Gibco BRL, Rockville, Md.) and placed into tissueculture flasks at 5×10⁶/ml in complete RPMI medium (containing 100 U/mlpenicillin/streptomycin, 10 mM HEPES buffer, 2 mM glutamine, 0.1 mMnon-essential amino acids; all from Gibco BRL); and 10% fetal calf serum(HyClone, Logan, Utah). After a 3 hours of incubation at 37° C. (5%CO₂), non-adherent cells were removed and the T cells were isolatedusing selection columns (R&D Systems, Minneapolis, Minn.). The adherentcells were washed with RPMI medium and incubated for 7 days in completeRPMI medium supplemented with 10 ng/ml IL-4 (R&D Systems) and 100 ng/mlGM-CSF (R&D systems). The non-adherent cells were then isolated, washed,and utilized as monocyte derived dendritic cells (mDCs) for allexperiments. The remaining adherent cells were removed usingtrypsin/EDTA and utilized in experiments employing adherent monocytes.

To determine whether the anti-CD40 antibodies of the inventionupregulate the expression of cell surface markers, the monocyte deriveddendritic cells were cultured with various concentrations of agonistantibodies for 48-72 hours, followed by staining (30 minutes, on ice, inthe dark) for HLA-DR, ICAM, B7-1, B7-2, CD40 and CD83, usingcommercially available fluorochrome labeled antibody reagents. The cellswere then analyzed on a FACS-Caliber (Becton-Dickinson).

The maximum fold increase of median fluorescence (at ≦1 μg/ml antibody),and mean EC₅₀ obtained using one of the anti-CD40 antibodies of theclaimed invention (21.4.1) are shown in Table 24.

TABLE 24 Upregulation of Dendritic Cell Surface Molecules by anAnti-CD40 Antibody of the Invention Maximum Fold Increase EC₅₀ (ng/ml)Mean +/− St. Dev. Mean +/− St. Dev. MHC II  7.7 +/− 5.6  252 +/− 353CD83 36.3 +/− 42.2  233 +/− 262 ICAM 10.4 +/− 4.8  241 +/− 140 B7-2 21.9+/− 9.4 71.4 +/− 44.4

We conducted similar experiments with B cells and mDCs using variousanti-CD40 antibodies of the invention and additional markers. Wemeasured the expression of B cell surface molecules (MHC-II, ICAM, B7-1,B7-2 and CD23) as described above but using 1 μg/ml of the anti-CD40antibody. The results of this experiment are presented in Table 25. Wemeasured the expression of dendritic cell surface molecules (MHC-II,ICAM, B7-1, B7-2 and CD83) after 72 hours as indicated above but using 1μg/ml of the anti-CD40 antibody. The results of this experiment arepresented in Table 26. Tables 25-26 show the fold increase in medianintensity +/− standard deviation.

TABLE 25 Upregulation of B-Cell Surface Molecules by Anti-CD40Antibodies Of The Invention MHC ICAM B7-1 B7-2 Class II (CD54) (CD 80)(CD86) CD23 B cell B cell B cell B cell B cell 3.1.1 3.2 +/− 2.6 1.3 +/−0.2 1.7 +/− 0.2 1.2 +/− 0.4 5.6 +/− 4.8 21.2.1 1.2 +/− 0.2 1.3 +/− 0.90.9 +/− 0.5  1.0 +/− 0.04 1.0 +/− 0.1 21.4.1 3.6 +/− 3.0 5.0 +/− 3.0 1.9+/− 0.8 1.8 +/− 0.7 21.5 +/− 34.8 22.1.1 1.4 +/− 0.5 1.1 +/− 0.2 1.2 +/−0.3 1.0 +/− 0.1 1.3 +/− 0.2 23.5.1 1.4 +/− 0.5 1.1 +/− 0.2 1.4 +/− 0.61.0 +/− 0.1 1.1 +/− 0.2 23.25.1 2.5 +/− 1.1 2.5 +/− 0.9 1.6 +/− 0.4 1.3+/− 0.2 4.3 +/− 2.3 23.28.1 1.1 +/− 0.2 1.1 +/− 0.2 1.8 +/− 0.6 1.0 +/−0.1 1.1 +/− 0.4 23.29.1 1.2 +/− 0.2 1.0 +/− 0.2 1.3 +/− 0.6 0.9 +/− 0.21.1 +/− 0.1 24.2.1 1.8 +/− 1.0 1.6 +/− 0.8 1.1 +/− 0.4 1.1 +/− 0.2 0.9+/− 0.6

TABLE 26 Upregulation of Dendritic Cell Surface Molecules by Anti-CD40Antibodies Of The Invention MHC Class ICAM B7-1 B7-2 II (CD54) (CD 80)(CD86) CD83 DC DC DC DC DC 3.1.1 4.4 +/− 2.4 1.5 +/− 0.7 1.8 +/− 0.923.7 +/− 33.5 15.2 +/− 18.2 21.2.1 1.8 +/− 1.3 1.5 +/− 0.9 0.9 +/− 0.4 7.4 +/− 10.5 10.8 +/− 16.5 21.4.1 5.0 +/− 3.8 3.7 +/− 1.4 1.5 +/− 1.112.9 +/− 13.3 48.6 +/− 49.5 22.1.1 2.3 +/− 1.2 1.6 +/− 0.7 1.4 +/− 1.016.3 +/− 25.5 12.0 +/− 17.0 23.5.1 2.3 +/− 1.8 1.2 +/− 0.5 1.1 +/− 0.610.7 +/− 17.5  9.2 +/− 11.1 23.25.1 2.1 +/− 1.8 2.4 +/− 1.0 1.1 +/− 0.53.3 +/− 4.2 13.6 +/− 28.9 23.28.1 2.4 +/− 1.7 2.7 +/− 2.1 1.3 +/− 0.610.6 +/− 17.5 18.3 +/− 22.6 23.29.1 2.0 +/− 1.5 1.2 +/− 0.4 0.9 +/− 0.5 8.4 +/− 10.6 10.6 +/− 13.1 24.2.1 4.7 +/− 3.0 2.1 +/− 1.2 3.8 +/− 3.856.6 +/− 95.8 31.2 +/− 28.4

Table 27 compares the upregulation of cell surface molecules indendritic cells over B cells in terms of the ratio of the mean-foldincrease on dendritic cells over the mean-fold increase on B cells.

TABLE 27 Upregulation of Cell Surface Molecules On Dendritic Cells OverB Cells B7-1 (CD80) B7-2 (CD86) MHC Class II ICAM (CD54)  3.1.1 1.0819.40 1.38 1.15 21.2.1 1.01 7.37 1.49 1.12 21.4.1 0.77 7.04 1.37 0.7422.1.1 1.18 16.36 1.61 1.44 23.5.1 0.83 10.54 1.59 1.06 23.25.1 0.662.57 0.85 0.98 23.28.1 0.71 10.81 2.16 2.57 23.29.1 0.73 9.07 1.66 1.2324.2.1 3.48 52.30 2.64 1.35

EXAMPLE VIII Enhancement of Cytokine Secretion

We conducted a monocyte derived dendritic cell assay to determinewhether the human anti-CD40 antibodies of the invention enhance thesecretion of IL-12p40, IL-12p70 and IL-8.

The monocyte derived dendritic cells and the adherent monocytes wereprepared as described above. Cells were cultured in the presence of ananti-CD40 antibody of the invention (21.4.1) or with a anti-keyholelimpet hemocyanin (KLH) antibody as a negative control. The cytokineswere measured in the supernatants at 24 hours by ELISA (R&D Systems). Insome studies (see Table 28), the monocyte derived dendritic cellstreated with the antibody also were co-stimulated with either 100 ng/mlLPS (Sigma), 1000 U/ml IFNγ (R&D Systems) or 25 ng/ml IL-1β R&D systems.

The anti-CD40 antibody enhanced IL-12p40, IL-12p70 and IL-8 productionin both monocyte derived dendritic cells and adherent monocytes. Thepresence of LPS further enhanced the production of IL-12p40 andIL-12p70. Only minimal levels of cytokines were detected in thesupernatants of dendritic cells incubated with the isotype controlantibody, anti-KLH. Representative results are presented in Table 28 andin FIGS. 3 and 4. Table 28 summarizes the principle cytokines producedby dendritic cells or adherent monocytes by 1 μg/ml of an anti-CD40antibody of the invention (21.4.1)+/−100 ng/ml LPS. As shown in FIG. 3,the anti-CD40 antibody enhanced IL-12p40 production by human dendriticcells. FIG. 4 illustrates enhanced IL-12p70 production by humandendritic cells in the presence of antibody and 100 ng/ml LPS.

TABLE 28 Enhancement of IL-12p40, IL-12p70 and IL-8 Secretion by anAnti-CD40 Antibody of the Invention Treatment LPS Induced cytokineAntibody 100 IL-12p40 IL-12p70 IL-8 Cell Type 1 μg/ml ng/ml pg/ml pg/mlpg/ml Dendritic 21.4.1 + 32252 1000 ND cell 21.4.1 −  1200  76 1200anti-KLH + 14280  352 ND anti-KLH −  200   4  150 Adherent 21.4.1 − NDND 7000 monocyte 21.4.1 + ND  425 ND anti-KLH − ND ND  400 anti-KLH + ND 30 ND ND = not determined

Similar experiments were performed using multiple anti-CD40 antibodiesof the invention. The monocyte derived dendritic cells were prepared asdescribed above and cultured in the presence of various concentrationsof the anti-CD40 antibodies and were co-stimulated with 100 ng/ml LPS(Sigma). The IL-12p70 in the supernatant was measured at 24 hours byELISA (R&D Systems) and the for each antibody EC₅₀ was determined. Theresults of the experiments are presented in Table 29.

TABLE 29 Enhancement of IL-12p70 Secretion In Dendritic Cells DCIL-12p70 Antibody Clone EC₅₀ μg/ml Max pg/ml 21.4.1 0.3 1796–7004 22.1.10.1  720–1040 23.25.1 0.2 540–960 23.5.1 0.1  676–1112 24.2.1 0.2 754–3680  3.1.1 0.2 668–960 23.28.1 0.2 1332–1404 23.29.1 0.1 852–90021.2.1 0.03 656–872

We also tested the ability of the anti-CD40 antibodies of the inventionto enhance the secretion of IFN-gamma from T cells in an allogenic Tcell/dendritic cell assay. To perform this assay, T cells and monocyteswere isolated from the peripheral blood of healthy volunteers. Monocyteswere differentiated into dendritic cells using the above-describedmethods. 1×10⁵ T cells obtained from an individual were cultured with1×10⁵ dendritic cells obtained from a different individual in thepresence of an anti-CD40 antibody of the invention or a controlantibody. After 4 days of culture, the supernatants were assayed forIFN-gamma secretion by ELISA. The results of this assay are shown inTable 30.

TABLE 30 Enhancement of IFN-gamma Secretion by Anti-CD40 Antibodies OfThe Invention Allo DC/T IFN_(γ) Antibody Clone EC₅₀ μg/ml Max pg/ml21.4.1 0.3 212 22.1.1 0.3 110–180 23.25.1 0.3 180–232 23.5.1 0.2 150–24024.2.1 0.2 111–194  3.1.1 0.1 100–195 23.28.1 0.2 120–190 23.29.1 0.3134–150 21.2.1 0.03 230–256

EXAMPLE IX Induction of Inflammatory Cytokines by the Anti-CD40Antibodies of the Invention

Antibodies 10.8.3, 15.1.1, 21.4.1 and 3.1.1 were tested in a whole-bloodcytokine release assay described by Wing et al., Therapeutic. Immunol.2:183-90 (1995) to determine if inflammatory cytokines are induced bythe antibodies at 1, and 100 μg/ml concentration. No significant releaseof TNF-α, IL-1β, IFN-γ, or IL-6 was observed with these antibodies atthe indicated concentrations in blood from 10 normal donors.

EXAMPLE X Enhancement of Immunogenicity of Cell Line Jy by Anti-CD40Antibodies

CD40 positive JIYOYE cells (ATCC CCL 87) (“Jy cells”) were cultured andmaintained in RPMI medium. JIYOYE cells were incubated for 24 hours withan anti-CD40 antibody of the invention (21.4.1), or with an isotypematched antibody (anti-KLH), in complete RPMI medium. Cells were thenwashed and treated with 25 mg mitomycin C (Sigma)/7 ml media for 60 min.These cells were then incubated with isolated human T cells at a 1:100ratio for 6 days at 37° C. (5% CO₂). T cells were then collected,washed, and the level of CTL activity determined against fresh chromium51 (New England Nuclear, Boston, Mass.) labeled JIYOYE cells. SpecificCTL activity was calculated as % specific cytolysis=(cytolysis Jy(cpm)−spontaneous cytolysis (cpm))/(total cytolysis (cpm)−spontaneouscytolysis (cpm)).

As FIG. 5 illustrates, an anti-CD40 antibody of the invention (21.4.1)significantly enhanced the immunogenicity against Jy cells treated withthe antibody.

EXAMPLE XI Animal Tumor Model

To further investigate the anti-tumor activity of the anti-CD40antibodies made in accordance with the invention, we designed aSCID-beige mouse model to test the in vivo effect of the antibody ontumor growth.

We obtained SCID-beige mice from Charles River and we allowed the miceto acclimate one week prior to use. We injected tumor cells (Daudi cells(ATCC CCL 213), CD40(−) K₅₆₂ cells (ATCC CCL 243) and CD40(+) Raji cells(ATCC CCL 86), BT474 breast cancer cells (ATCC HTB 20) or PC-3 prostatecells (ATCC CRL 1435)) subcutaneously at a concentration of 1×10⁷cells/animal. In some cases, we injected T cells (5×10⁵) and dendriticcells (1×10⁵) from the same human donor along with the tumor cells. Wealso injected an anti-CD40 antibody of the invention, or an isotypematched control (anti-KLH), intraperitoneally, immediately prior totumor injection (one injection only). We then measured tumor growth.Specific experiments are described below.

In one experiment, we injected an anti-CD40 antibody of the invention(21.4.1), or an isotype matched control (anti-KLH), intraperitoneally,at a dose of 10 mg/kg immediately prior to tumor injection (oneinjection only). The tumor cells (Daudi cells) were injectedsubcutaneously at a concentration of 1×10⁷ cells/animal. We measuredtumor growth with calipers at days 17, 19, 20, 21, 25, 26, 27 and 28after implantation in the presence of human T cells and dendritic cells.As shown in FIG. 6, the anti-CD40 antibody inhibited tumor growth byabout [60]%.

In another experiment, we injected an anti-CD40 antibody of theinvention (21.4.1), or an isotype matched control (anti-KLH),intraperitoneally, at a dose of 0.1 mg/kg, 1 mg/kg or 10 mg/kgimmediately prior to tumor injection (one injection only). The tumorcells (K562 cells) were injected subcutaneously at a concentration of1×10⁷ cells/animal. In this experiment we injected T cells (5×10⁵) anddendritic cells (1×10⁵) from the same human donor along with the tumorcells. We measured tumor growth with calipers at days 17, 19, 20, 21,25, 26, 27 and 28 after implantation. As shown in FIG. 7, the anti-CD40antibody inhibited tumor growth by 60-85%.

In another experiment, we injected an anti-CD40 antibody of theinvention (21.4.1, 23.29.1 or 3.1.1), or an isotype matched control(anti-KLH), intraperitoneally, immediately prior to tumor injection (oneinjection only). The isotype matched control antibody and antibody21.4.1 were injected at a dose of 1 mg/ml. Antibodies 23.29.1 and 3.1.1were injected at a dose of 1, 0.1, 0.01, 0.001 or 0.0001 mg/kg. Thetumor cells (K562 cells) were injected subcutaneously at a concentrationof 1×10⁷ cells/animal. In this experiment we injected T cells (5×10⁵)and dendritic cells (1×10⁵) from the same human donor along with thetumor cells. We then measured tumor growth with calipers at day 28 afterimplantation. The results of this experiment are shown in FIGS. 8 and 9.Each point in the figures represents a measurement from an individualanimal.

In another experiment, we injected an anti-CD40 antibody of theinvention (21.4.1), or an isotype matched control (anti-KLH),intraperitoneally, immediately prior to tumor injection (one injectiononly). The antibodies were injected at a dose of 1, 0.1, 0.01, 0.001 or0.0001 mg/kg. The tumor cells (Raji cells) were injected subcutaneouslyat a concentration of 1×10⁷ cells/animal. In some animals, we injected Tcells (5×10⁵) and dendritic cells (1×10⁵) from the same human donoralong with the tumor cells. We then measured tumor growth with calipersat day 28 after implantation. The results of this experiment are shownin FIG. 10. Each point in the figure represents a measurement from anindividual animal.

In yet another experiment, we injected an anti-CD40 antibody of theinvention (21.4.1, 23.28.1, 3.1.1 or 23.5.1), or an isotype matchedcontrol (anti-KLH), intraperitoneally, immediately prior to tumorinjection (one injection only). The antibodies were injected at a doseof 1 or 0.1 mg/kg. The tumor cells (Raji cells) were injectedsubcutaneously at a concentration of 1×10⁷ cells/animal. We thenmeasured tumor growth with calipers at day 28 after implantation. Theresults of this experiment are shown in FIG. 11. Each point in thefigure represents a measurement from an individual animal.

In yet another experiment, we injected an anti-CD40 antibody of theinvention (21.4.1, 23.29.1, or 3.1.1), or an isotype matched control(anti-KLH), intraperitoneally, immediately prior to tumor injection (oneinjection only). The antibodies were injected at a dose of 1 mg/kg. Thetumor cells (BT474 breast cancer cells) were injected subcutaneously ata concentration of 1×10⁷ cells/animal. We injected T cells (5×10⁵) anddendritic cells (1×10⁵) from the same human donor along with the tumorcells. We then measured tumor growth with calipers at day 39 afterimplantation. As shown in FIG. 12, all of the antibodies inhibitedbreast cancer tumor growth. Each point in the figure represents ameasurement from an individual animal.

In yet another experiment, we injected an anti-CD40 antibody of theinvention (3.1.1), or an isotype matched control (anti-KLH),intraperitoneally, immediately prior to tumor injection (one injectiononly). The antibodies were injected at a dose of 1 mg/kg. The tumorcells (PC-3 prostate tumor cells) were injected subcutaneously at aconcentration of 1×10⁷ cells/animal. We then measured tumor growth withcalipers at day 41 after implantation. As shown in FIG. 13, theanti-CD40 antibody inhibited prostate tumor growth by about 60%. Eachpoint in the figure represents a measurement from an individual animal.

EXAMPLE XII Survival of SCID-Beige Mice Injected with Daudi Tumor Cellsand Treated with the Anti-CD40 Antibodies of the Invention

In another experiment, we injected an anti-CD40 antibody of theinvention, or an isotype matched (one injection) control,intraperitoneally, immediately prior to tumor injection. The antibodieswere injected at a dose of 1 or 0.1 mg/kg. The tumor cells (Daudi cells)were injected intravenously at a dose of 5×10⁶ cells/animal. We thenmonitor animal survival. As shown in FIG. 14, all of the anti-CD40antibodies tested prolonged the survival of mice injected tumors by atleast six days.

Table 31 lists the ED₅₀ of the anti-CD40 antibodies in the differentsolid tumor models described in Example XI. Table 31 summarizes the invivo anti-tumor activity of some of the anti-CD40 antibodies of theinvention in SCID mice. In addition, the table lists the ED₅₀ of theanti-CD40 antibodies in the Daudi systemic tumor model described abovein Example XII.

TABLE 31 ED₅₀ Of Anti-CD40 Antibodies Of The Invention Using DifferentIn Vivo Tumor Models in SCID mice CD40(−) K562 CD40(+) Raji & T/DC &T/DC CD40(+) Raji CD40(+) sub- sub- sub- Daudi cutaneous cutaneouscutaneous intra-venous Antibody (mg/kg) (mg/kg) (mg/kg) (mg/kg) 21.4.10.005 0.0008 0.016 0.1 22.1.1 0.01 ND >1.0 0.1 23.25.1 ≧1.0 ND >1.0 ND23.5.1 >1.0 ND ≧1.0 ND 24.2.1 >1.0 ND >1.0 ND  3.1.1 0.02 ND ≧0.1 ≦0.123.28.1 >1.0 ND ≧1.0 0.1 23.29.1 0.009 ND >1.0 ≦0.1 21.2.1 ≦1.0 ND ND NDND = Not Done

EXAMPLE XIII Determination of Affinity Constants (K_(D)) of Fully HumanAnti-CD40 Antibodies by BIAcore

We performed affinity measures of purified antibodies by surface plasmonresonance using the BIAcore 3000 instrument, following themanufacturer's protocols.

The Biosensor biospecific interaction analysis instrument (BIAcore) usessurface plasmon resonance to measure molecular interactions on a CM5sensor chip. Changes in the refractive indices between two media, glassand carboxymethylated dextran, caused by the interaction of molecules tothe dextran side of the sensor chip, is measured and reported as changesin arbitrary reflectance units (RU) as detailed in the manufacturer'sapplication notes.

The carboxymethylated dextran surface of a flow cell on a sensor chipwas activated by derivatization with 0.05 M N-hydroxysuccinimidemediated by 0.2 M N-ethyl-N′-(dimethylaminopropyl) carbodiimide for 7min. CD40-Ig fusion protein (described in Example I) at a concentrationof 5 μg/ml, in 10 mM Na acetate, pH 3.5, was manually injected into theflow cell at a rate of 5 μl/min and covalently immobilized to the flowcell surface with the desired amount of RU's. Deactivation of unreactedN-hydroxysuccinimide esters was performed using 1 M ethanolaminehydrochloride, pH 8.5. Following immobilization, the flow cells arecleaned of any unreacted or poorly bound material with 5 regenerationinjections of 5 μl of 50 mM NaOH until a stable baseline is achieved.Flow cell 2, a high density surface, measured approximately 300 RU'sfollowing surface preparation and flow cell 3, a low density surface,measured approximately 150 RU's. For flow cell 1, the activated blanksurface, 35 μl of 10 mM Na acetate buffer was injected duringimmobilization in place of antigen. Flow cell 4 contained approximately450 RU's of immobilized CTLA4-Ig, an irrelevant antigen control.

A dilution series of each antibody was prepared in the concentrationrange of 100 μg/ml to 0.1 μg/ml by half logs. The flow rate was set at 5μl/min and 25 μl of each concentration point sample was injected overthe sensor chip with a regeneration injection of 5 μl of 50 mM NaOHbetween each concentration of antibody injected. The data was analyzedusing BIAevaluation 3.0 software.

In reverse orientation kinetic experiments, the antibody 21.4.1 wasimmobilized to the sensor chip surface using the protocol describedabove. Anti-KLH was used as a control antibody surface. The antigen,CD40-Ig fusion protein, was injected in the concentration range of 100μg/ml to 0.1 μg/ml.

Table 32 lists affinity measurements for representative anti-CD40antibodies of the present invention:

TABLE 32 Affinity Measurements For Anti-CD40 Antibodies Of The InventionAntibody K_(on) (1/Ms) K_(off) (1/s) K_(D) (M)  3.1.1 1.12 × 10⁶ 3.31 ×10⁻⁵ 3.95 × 10⁻¹¹ 10.8.3 2.22 × 10⁵ 4.48 × 10⁻⁷ 2.23 × 10⁻¹² 15.1.1 8.30× 10⁴ 2.83 × 10⁻⁷ 4.05 × 10⁻¹² 21.4.1 8.26 × 10⁴ 2.23 × 10⁻⁵ 3.48 ×10⁻¹⁰ 22.1.1 9.55 × 10⁵ 1.55 × 10⁻⁴ 2.79 × 10⁻¹⁰ 23.25.1 3.83 × 10⁵ 1.65× 10⁻⁷ 7.78 × 10⁻¹² 23.28.1 7.30 × 10⁵ 8.11 × 10⁻⁵ 1.61 × 10⁻¹⁰ 23.29.13.54 × 10⁵ 3.90 × 10⁻⁵ 7.04 × 10⁻¹¹

EXAMPLE XIV Epitope Mapping of Anti-CD40 Antibodies

The binding assays were done using Protein A purified CD40-human IgG1 Fcfusion antigen. The human CD40-IgG1 Fc fusion protein was cloned atPfizer. The human CD40-IgG1 fusion protein was expressed in a mammaliancell line and purified over Protein A column. The purity of the fusionantigen was assessed by SDS/PAGE.

CD40 has a structure of a typical type I transmembrane protein. Themature molecule is composed of 277 amino acids. The extracellular domainof CD40 consists of four TNFR-like cysteine rich domains. See, e.g.,Neismith and Sprang, TIBS 23:74-79 (1998); van Kooten and Banchereau, J.Leukocyte Biol. 67:2-17 (2000); Stamenkovic et al., EMBO J.8:1403-1410(1989).

Binding of Anti-CD40 Antibodies to Reduced and Non-Reduced Human CD40:

Because the extracellular domain of CD40 consists of four cysteine richdomains, disruption of the intramolecular bonds, by reducing agent, canchange antibody reactivity. To determine whether disruption of theintramolecular bonds, by reducing agent, changed the reactivity ofselected anti-CD40 antibodies of the invention, purified CD40-hIgG wasloaded on SDS/PAGE (4-20% gel) under non-reducing (NR), or reducing (R),conditions. SDS/PAGE was performed by the method of Laemmli, using amini-gel system. Separated proteins were transferred on tonitrocellulose membrane. Membranes were blocked using PBS containing 5%(w/v) non fat dried milk for at least 1 hour before developing, andprobed for 1 hr with each antibody. Anti-CD40 antibodies were detectedusing HRP-conjugated goat anti-human immunoglobulins (1:8,000 dilution;Catalog No. A-8667 from Sigma). Membranes were developed by usingenhanced Chemiluminescence (ECL®; Amersham Bioscience) according to themanufacturer's instructions.

The Western Blot was then probed with four anti-CD40 antibodies of theinvention: 21.4.1, 23.25.1, 23.29.1 and 24.2.1 (1 μg/ml,) followed byHRP conjugated goat anti-human IgG (1:8000 dilution). The results ofthis experiment are show in FIG. 15. The results indicate thatantibodies 21.4.1, 23.25.1, 23.29.1 and 24.2.1 bind non-reduced but donot bind reduced CD40, the antibodies, thus, recognize a conformationalepitope.

Binding of Anti-CD40 Antibodies to Human CD40 Domain Deleted Proteins:

The extracellular region of CD40 includes four TNFR-like repeat domains(referred to as D1-D4). See, e.g., Neismith and Sprang, TIBS 23:74-79(1998); van Kooten and Banchereau, J. Leukocyte Biol. 67:2-17 (2000);Stamenkovic et al., EMBO J. 8:1403-1410(1989). FIG. 16 shows the aminoacid sequences of the mouse and human CD40 domains D1-D4. To investigatethe contribution of different regions of the CD40 molecule in thepresentation of the epitope, a number of domain deleted mutants wereconstructed.

To make the human CD40 deletion constructs, the entire extracellulardomain of human CD40 (amino acids 1-193) was amplified from human Bcells (CD19+) cDNA (Multiple tissue cDNA panels, Catalog No. K1428-1,from Clontech) by PCR using sequence specific primers, and a 6×His-tagwas added at the C-terminal. A human CD405′ primer 5′-GCAAGCTTCACCAATGGTTCGTCTGCCTCTGCAGTG-3′ (SEQ ID NO: 135) was used with differentcombination of 3′ primers for cloning of full length and truncated CD40molecules. The 3′ primer for cloning the full-length extracellulardomain of human CD40 was: 5′-TCAGTGATGGTGATGGTGATGTCTCAGCCGATCCTGGGGACCA-3′ (SEQ ID NO: 136). The 3′ primer used to clone the D1-D3domains of human CD40 was: 5′-TCAGTGATGGTGATGGTGATGTGGGCAGGGCTCGCGATGGTAT-3′ (SEQ ID NO: 137) The 3′ primer used to clone theD1-D2 domains of CD40 was: 5′-TCAGTGATGGTGATGGTGATGACAGGTGCAGATGGTGTCTGTT-3′ (SEQ ID NO: 138). After these constructs oftruncated CD40 cDNA were generated, they were expressed in the 293F cellline using the pCR3.1 vector (Invitrogen). The CD40-6×H is fusionproteins (SEQ ID NOS 139-141) were purified by elution from a nickelcolumn.

The amino acid sequences of these four deletion mutants are shown inTable 33.

TABLE 33 CD40 His-Tag Fusion Proteins Deletion Mutant: Amino AcidSequence (leader sequence underlined) Human CD40-6XHisMVRLPLQCVLWGCLLTAVHPEPPTACREKQYLNS (SEQ ID NO:139) (full lengthextra-cellular domain)- QCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGT SETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQJATGVSDTICEPCPVGFFSNVSSAFEK CHPWTSCETKDLVVQQAGTNKTDVVCGPQDRHHHHHH Human CD40 (D1-D3)-6xHis MVRLPLOCVLWGCLLTAVHPEPPTACREKQYLINS(SEQ ID NO:140) QCCSLCQPGQKIVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGT SETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPHHHHHH Human CD40 (D1-D2)-6XhisMVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINS (SEQ ID NO:141)QCCSLCQPGQKLVSDCTEFTETECLPC GESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCHHHHHH

To express these human CD40 deletion constructs, the constructs werecloned into the pCR3.1 vector (Invitrogen) and expression was assessedin various stable and transiently transfected 293F cell lines. Thesupernatants from transiently transfected 293F cells were analyzed forbinding to antibodies 21.4.1, 23.25.1, 23.29.1 and 24.2.1 by ELISA andWestern Blot.

ELISA assays were preformed using supernatant from 293F cellstransfected with different CD40 constructs. ELISA plates were coatedgoat anti-human CD40 polyclonal antibodies (R&D catalog No. AF 632) orgoat anti-mouse CD40 polyclonal antibodies (R&D catalog No. AF 440)diluted to 1 μg/ml in ELISA plate coating buffer. Expression of CD40constructs in 293F cells was confirmed by detection with biotinylatedgoat anti-human CD40 (R&D catalog No. BAF 632), goat anti-mouse CD40(R&D catalog No. BAF 440), or HRP-conjugated anti-H is (C terminal)antibody (Invitrogen, Catalog No. 46-0707). Binding of anti-CD40 humanantibodies were detected with HRP conjugated goat anti-human IgG (FCspecific Caltag H10507), diluted 1:2,000. The results, as shown in Table34, indicate that most if not all of the epitope recognized by mAbs21.4.1, 23.28.1 and 23.29.1 is located in the D1-D2 region of CD40 whilethe epitope for mAb 24.2.1 is located at least partly in domain D3-D4. Ahuman CD40-rabbit Fc fusion protein was used a control to confirm thespecificity of the antibody binding.

TABLE 34 ELISA: Antibody Binding To CD40 Deletion Mutants Human CD40Human CD40 (D1-D2)-6Xhis (D1-D3)-6XHis Human CD40-6XHis 21.4.1 + + +23.25.1 + + + 23.29.1 + + + 24.2.1 − + + anti-His + + + anti-RbIg ND NDND

The CD40 deletion constructs also were analyzed by Western Blotanalysis. The results are shown in Table 35. The ELISA results show thatthe binding site of antibodies 21.4.1, 23.25.1, 23.29.1 and 24.2.1involves domains D1-D3. The results also show that the binding site forantibodies 21.4.1, 23.25.1 and 23.29.1 involve domains D1-D2, and thatthe binding site of antibody 24.2.1 involves domain D3.

TABLE 35 Western Blot: Antibody Binding To CD40 Deletion Mutant HumanCD40(D1-D3)-6Xhis Human CD40-6Xhis 21.4.1 + + 23.25.1 + + 23.29.1 + +24.2.1 + + anti-His + + Anti-RbIg ND NDBinding of Anti-CD40 Antibodies to Mouse CD40:

We set out to determine the ability of antibodies 21.4.1, 23.25.1,23.29.1 and 24.2.1 to bind mouse CD40.

For this experiment, mouse CD40 was amplified from mouse B cells cDNA.Mouse CD40(D1-D3)-6×H is fusion protein was cloned into pCR3.1, whichutilizes the CMV promoter, to drive transcription. The 5′ primer used toclone the extracellular domain of the mouse CD40 was:5′-TGCAAGCTTCACCATGGTGTCTTTGCCTCGGCTGTG-3′ (SEQ ID NO: 146). The 3′primer used to clone the D1-D3 domains of mouse CD40 was:5′-GTCCTCGAGTCAGTGATGGTGATGGTGATGTGGGCAGGGATGACAGAC-3′ (SEQ ID NO: 147).Mouse and human cDNA constructs were transfected into 293F cellstransiently. The expression of recombinant CD40 was detected by ELISAusing polyclonal antibodies against mouse and human CD40, anti-H isantibodies, and anti-CD40 antibodies 21.4.1, 23.25.1, 23.29.1 and24.2.1. The results of these experiments are shown in Table 36. Thisexperiment shows that all antibodies are specific to human CD40 and donot cross react with mouse CD40.

TABLE 36 Cross-Reactivity of Mouse and Human CD40 Mouse CD40(D1-D3)-Human CD40(D1-D3)- 6Xhis 6XHis 21.4.1 No Yes 23.25.1 No Yes 23.29.1 NoYes 24.2.1 No Yes goat anti-human CD40 No Yes goat anti-mouse CD40 YesNo Anti-His Yes YesBinding of Anti-CD40 Antibodies to of Human/Mouse Chimeric CD40:

Because antibodies 21.4.1, 23.25.1, 23.29.1 and 24.2.1 do not bind mouseCD40, we constructed human/mouse chimeric CD40 proteins to moredefinitively map the epitopes of those antibodies.

For the construction of in-frame fusions of the human and murine CD40chimeric proteins, we used unique restriction sites at the borders ofCD40 domains at identical positions in the cDNA of both human and mouseCD40. Various cDNA constructs of CD40 were generated using the EcoRIrestriction site at the end of domain 1 (nucleotide 244, amino acid 64)and the BanI restriction site at the end of domain 2 (nucleotide 330,amino acid 94) (FIG. 17).

Various CD40 domains were amplified by PCR and ligated. This approachallowed the replacement of various domains of the mouse CD40 by thehomologous domains from the human CD40. The constructs obtained areshown in FIG. 18.

We then determined whether antibodies 21.4.1, 23.25.1, 23.29.1 and24.2.1 were able to bind the mouse/human chimeric CD40 proteins byELISA. The results of this experiment are shown in Table 37. As shown inTable 37, mAbs 21.4.1 and 23.25.1 recognize and epitope that is locatedpartly in D1 and partly in D2; mAb 23.29.1 recognizes an epitope locatedmostly if not completely in D2; and mAb 24.2.1 recognizes an epitopelocated in D2 and D3.

TABLE 37 Antibody Binding to Chimeric CD40 Proteins HuD1, HuD2, HuD1,Antibody HuD1 HuD2 HuD3 D2 D3 D3 21.4.1 No No No Yes No No 23.25.1 No NoNo Yes No No 23.29.1 No Yes No Yes Yes No 24.2.1 No No No No Yes No

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the invention.

1. A monoclonal antibody or an antigen-binding portion thereof thatspecifically binds to and activates human CD40, wherein said antibody orportion comprises: (a) a heavy chain CDR1 amino acid sequence, CDR2amino acid sequence, and CDR3 amino acid sequence, respectively, ofamino acid residues 26-35, 50-66, and 99-115 of SEQ ID NO: 42; and (b) alight chain CDR1 amino acid sequence, CDR2 amino acid sequence, and CDR3amino acid sequence, respectively, of amino acid residues 24-34, 50-56,and 89-97 of SEQ ID NO:
 44. 2. A monoclonal antibody or anantigen-binding portion thereof that specifically binds to and activateshuman CD40, wherein said antibody or portion comprises: (a) the lightchain variable region amino acid sequence of SEQ ID NO: 44; and (b) theheavy chain variable region amino acid sequence of SEQ ID NO:
 42. 3. Themonoclonal antibody or antigen-binding portion thereof according toclaim 1 or 2, wherein said antibody is an IgG molecule selected from thegroup consisting of an IgG1 subclass, an IgG2 subclass, and an IgG3subclass.
 4. The monoclonal antibody or antigen-binding portion thereofaccording to claim 3, wherein said antibody is an IgG2 subclass.
 5. Amonoclonal antibody or an antigen-binding portion thereof thatspecifically binds to and activates human CD40, wherein said antibody orportion comprises a light chain variable region amino acid sequencecomprising SEQ ID NO: 44 and a heavy chain variable region amino acidsequence comprising SEQ ID NO:
 42. 6. The monoclonal antibody accordingto claim 5, which is of the IgG2 subclass.
 7. The monoclonal antibodyaccording to claim 5, which is of the IgG1 subclass.
 8. A monoclonalantibody that specifically binds to and activates human CD40, whereinsaid antibody comprises a light chain amino acid sequence comprising SEQID NO: 48 without amino acid residues 1-20, and a heavy chain amino acidsequence comprising SEQ ID NO:46 without amino acid residues 1-19. 9.The monoclonal antibody according to claim 8, wherein said antibodycomprises a light chain with an amino acid sequence consisting of SEQ IDNO: 48 without amino acid residues 1-20, and a heavy chain with an aminoacid sequence consisting of SEQ ID NO: 46 without amino acid residues1-19.
 10. A monoclonal antibody having the heavy and light chain aminoacid sequences of the antibody produced by hybridoma 21.4.1 havingAmerican Type Culture Collection (ATCC) accession number PTA-3605.
 11. Amonoclonal antibody or an antigen-binding portion thereof thatspecifically binds to and activates human CD40, wherein said antibody orportion comprises: (a) a light chain variable region amino acid sequenceencoded by a nucleic acid comprising the sequence of SEQ ID NO: 43; and(b) a heavy chain variable region amino acid sequence encoded by anucleic acid comprising the sequence of SEQ ID NO:
 41. 12. A monoclonalantibody that specifically binds to and activates human CD40, whereinsaid antibody comprises: (a) a light chain amino acid sequence encodedby a nucleic acid comprising the sequence of SEQ ID NO: 47 withoutnucleotides 1-60; and (b) a heavy chain amino acid sequence encoded by anucleic acid comprising the sequence of SEQ ID NO: 45 withoutnucleotides 1-57.
 13. A pharmaceutical composition comprising themonoclonal antibody or antigen-binding portion thereof according toclaim 1 and a pharmaceutically acceptable carrier.
 14. A pharmaceuticalcomposition comprising the monoclonal antibody or antigen-bindingportion thereof according to claim 5 and a pharmaceutically acceptablecarrier.
 15. A pharmaceutical composition comprising the monoclonalantibody according to claim 8 or an antigen-binding portion thereof anda pharmaceutically acceptable carrier.
 16. A pharmaceutical compositioncomprising the monoclonal antibody or antigen-binding portion thereofaccording to claim 2 and a pharmaceutically acceptable carrier.
 17. Apharmaceutical composition comprising the monoclonal antibody accordingto claim 9 or an antigen-binding portion thereof and a pharmaceuticallyacceptable carrier.
 18. A pharmaceutical composition comprising themonoclonal antibody according to claim 10 or an antigen-binding portionthereof and a pharmaceutically acceptable carrier.
 19. A pharmaceuticalcomposition comprising the monoclonal antibody or antigen-bindingportion thereof according to claim 11 and a pharmaceutically acceptablecarrier.
 20. A pharmaceutical composition comprising the monoclonalantibody according to claim 12 or an antigen-binding portion thereof anda pharmaceutically acceptable carrier.
 21. A monoclonal antibody or anantigen-binding portion thereof comprising the CDR1, CDR2, and CDR3 ofthe heavy chain amino acid sequence and the CDR1, CDR2, and CDR3 of thelight chain amino acid sequence of the antibody produced by hybridoma21.4.1 having American Type Culture Collection (ATCC) accession numberPTA-3605.
 22. The monoclonal antibody or antigen-binding portion ofclaim 21, comprising the heavy chain variable region amino acid sequenceand the light chain variable region amino acid sequence of the antibodyproduced by said hybridoma.